LOCUS G00 backbone 124 bp ds-DNA linear UNA 16-APR-2016 DEFINITION G00 backbone ACCESSION . VERSION . KEYWORDS G00 backbone SOURCE natural DNA sequence ORGANISM unspecified REFERENCE 1 (bases 1 to 124) AUTHORS . TITLE Direct Submission JOURNAL Exported Tuesday, Jun 7, 2016 from SnapGene Viewer 3.1.2 http://www.snapgene.com FEATURES Location/Qualifiers source 1..124 /organism="unspecified" /mol_type="genomic DNA" promoter 6..24 /note="T7 promoter" /note="promoter for bacteriophage T7 RNA polymerase" gap 25..44 /estimated_length="20" misc_RNA 45..120 /note="gRNA scaffold" /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" ORIGIN 1 gaaattaata cgactcacta taggnnnnnn nnnnnnnnnn nnnngtttta gagctagaaa 61 tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc 121 tttt // LOCUS pLENT-PF16-YFPTY_Var4.4 6350 bp ds-DNA circular SYN 06-DEC-2016 DEFINITION pLENT-PF16-YFPTY_Var4.4 ACCESSION . VERSION . KEYWORDS pLENT-PF16-YFPTY Var4.4 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6350) AUTHORS . TITLE Direct Submission JOURNAL Exported Tuesday, Dec 6, 2016 from SnapGene Viewer 3.1.4 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6350 /organism="synthetic DNA construct" /mol_type="other DNA" misc_feature 6..23 /note="Untitled" CDS 8..1264 /codon_start=1 /note="PF16-eYFP-TY" /translation="EKEPEDHIRAAAAWSLGQLGRHSADHAKAVADCNVLPRLLDVYLS PSSSDDLQTKSKRALKAIIQHCVYLPALEPLLHPDAPQDVLKYVCGQYAKVLPTDVAAK REFVANRGLATVQRIKAEPGSSLAESIQIINSCFPPEIVEYYSPEYAQTFIEKIENYHV QQHTSVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLP VPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVK FEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIE DGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLEFVTAAGITL GMDELYKSREVHTNQDPLD" misc_feature 146..163 /note="Untitled" misc_feature 255..272 /note="Untitled" misc_feature 326..343 /note="Untitled" misc_feature 372..389 /note="Untitled" misc_feature 411..428 /note="Untitled" misc_feature 441..458 /note="Untitled" misc_feature 458..475 /note="Untitled" misc_feature 473..490 /note="Untitled" misc_feature 482..499 /note="Untitled" misc_feature 512..1225 /note="Untitled" CDS 512..1225 /codon_start=1 /note="eYFP" /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK FICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLE FVTAAGITLGMDELYK" misc_feature 1226..1225 /note="Untitled" CDS 1232..1261 /codon_start=1 /note="TY" /translation="EVHTNQDPLD" misc_feature 1266..1806 /note="pJ1170 (pLEnT-YB) LmxM.34.1670 -.1660 intergenic" /note="Promega Vector: Taq makes single A overhangs, PCR products can be directly cloned into pGEM-t without any digest!" misc_feature 1811..2203 /note="pLENT-PF16-YFPTY Var4.3.gcs" CDS 1811..2203 /codon_start=1 /note="BlastR" /translation="MPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTGVN VYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGIKA IVKDSDGQPTAVGIRELLPSGYVWEG" misc_feature 2210..2695 /note="CfGSPS 3'" misc_feature 2691..3196 /note="pLENT-PF16-GFPTY Var2" misc_feature 2696..3195 /note="Untitled" misc_feature complement(2702..2719) /note="Untitled" misc_feature complement(2711..2728) /note="Untitled" misc_feature complement(2726..2743) /note="Untitled" misc_feature complement(2743..2760) /note="Untitled" misc_feature complement(2773..2790) /note="Untitled" misc_feature complement(2812..2829) /note="Untitled" misc_feature complement(2858..2875) /note="Untitled" misc_feature complement(2929..2946) /note="Untitled" misc_feature complement(3038..3055) /note="Untitled" misc_feature complement(3178..3195) /note="Untitled" CDS complement(4894..5751) /codon_start=1 /note="Amp" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" ORIGIN 1 agctttggag aaagagcccg aagaccacat acgtgccgcc gcggcgtggt cactggggca 61 gctcggccgc catagcgccg atcacgccaa ggcggtggcg gactgcaacg tgcttcctcg 121 tctgttggac gtgtacctca gccccagcag ctccgacgac ctgcagacga agagcaagcg 181 cgccctcaag gctatcatcc agcactgtgt ctacctaccc gccctagaac cgctgctgca 241 ccccgatgcg ccgcaggacg tcctcaagta cgtctgcggt cagtatgcga aggtgctgcc 301 caccgacgtg gccgcgaagc gcgagttcgt ggcgaaccgc ggcttggcca cggtgcagcg 361 catcaaggct gagccgggca gttccttggc cgagtccatc cagatcatca acagctgctt 421 cccgccggag attgtagaat actattcgcc agagtacgca cagacgttta tcgagaagat 481 cgagaactac cacgtgcagc agcacactag tgtgagcaag ggcgaggagc tgttcaccgg 541 ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc 601 cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac 661 cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ttcggctacg gcctgcagtg 721 cttcgcccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga 781 aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc 841 cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt 901 caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt 961 ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa 1021 catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga 1081 cggccccgtg ctgctgcccg acaaccacta cctgagctac cagtccgccc tgagcaaaga 1141 ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac 1201 tctcggcatg gacgagctgt acaagtctag agaggtccat actaaccaag atccacttga 1261 ctaaggatcc agatctcgcc gaacacgatc gaggctcagt gaagcacaag cgtgtcacga 1321 tgggtgcgtg cgcacgtgta ggccacttcc gatgtgcgca catccatcac cggagcgcgt 1381 ctttctcctt tatcatcttc tttgttcttc tttccgtttt taccgtttca tgaaaaaaaa 1441 gatttgaagc gtgcgactcc aaccgggacg tccccctccc ttcccctcct cccccgcagc 1501 gcctcctttt ccttctgtgt gtatatgggg ggtttggatc ttagtggcac ggcctgctca 1561 tcgactgtct gtctgtctgt ctgtctgtct ctgtctcggc gccatcagtc ttttgctacc 1621 accctctccc tactactcct cctctctgta gcacccatac gtcgcgatca acgcgaaatc 1681 cacgtctagg ccgcaccccg ctcgatgtgg cgccgtcaca ggtgtgttgc cgtcatctga 1741 gtagggggtg gcaagtgaca ccgtcgcata agacgcctgg tacacacaca cacacttcag 1801 tcatagaatt atgcctttgt ctcaagaaga atccaccctc attgaaagag caacggctac 1861 aatcaacagc atccccatct ctgaagacta cagcgtcgcc agcgcagctc tctctagcga 1921 cggccgcatc ttcactggtg tcaatgtata tcattttact gggggacctt gtgcagaact 1981 cgtggtgctg ggcactgctg ctgctgcggc agctggcaac ctgacttgta tcgtcgcgat 2041 cggaaatgag aacaggggca tcttgagccc ctgcggacgg tgccgacagg tgcttctcga 2101 tctgcatcct gggatcaaag ccatagtgaa ggacagtgat ggacagccga cggcagttgg 2161 gattcgtgaa ttgctgccct ctggttatgt gtgggagggc taaccatgga gatccagcag 2221 gcggagaaag agggaagtat aaggcggacg cataggtcgg agtatcgaga aaaagaggca 2281 gagatgggtg gtggcggagc gcccctctct gcccttctct gttttactgt tcccgccacg 2341 tcgccaactc ctctttttgt ttgctctaat gtcggtgcta tcctcttcct ctttttactc 2401 tccgtttttc ttccttccgt tttgtcttgt tatcaccgtt atttttcttc ttctttctta 2461 gccgatttgg gtctcctgcc tacggcagcg gtgatgagtc gcacttccgt ccccctttcc 2521 tctccgtaag tactccctcg atgcctcagg cgcttctatt ttgcggcact gtgctgacca 2581 cctcccacgt gtgcagtgag agcgccagag acattcagga gaagagggaa gaggggaagt 2641 aaataccaaa gcgaggaaga tgtctttctc gctgcttctc atcctgttga ccggtgcggg 2701 ggaggaggcg tcgcggatgc tcagcgggcc tttcggcaca gtcacgccca tgcacgctgc 2761 tcgtcgcgca cgccctcttg gctttcagtg ttcctccggt cattgcctgc tctgctgtgt 2821 cgtcggtaca cgccgtcgtc atcgaagtgc cccccttctt cgctgcacat tactgtttat 2881 atacacgtat gcatgtgttg tccgccacca tagagttggc gactcttctg tgttgttggg 2941 ttggccgttg acgcttgtgt tttgtgttgg agtgcgtctg tgtgatgatg aaggagaggg 3001 aggaggagtg gtgatccttc cctcccctcc cctcccctcc cctccccctt gttgccacaa 3061 tgtacatata catgatggat ataaaaagag cgagaggcag cgagagagag agtgccaaga 3121 ggcgcgtggc ctttaccgct tccctgagtc gttctttcgc ctctcctggc attggcgtct 3181 cgcgaagagg cttggcgtac gttttactat atacatattt gtttctgtgc tgatgccatt 3241 tgtccgctac tccatatcgt gcgtgtttgt tttccagtgg tgacgccttc cgcatcattt 3301 taatcaattc gaggcgaaaa gggtgatttg agtgttggag tgggcttttt tatttatttg 3361 actccaaaca tcttttcttt tcccggtatc ttcgggtccg tcacgcaggt cggtgggtgt 3421 gtccgagtgc gcttctttac tcacgtgccg ctgcgcacat acacagattt taaaagcacg 3481 catacacgat ctgtgccttc aaaactaatc aacaaaaaca aaaatacaaa aaagacaaga 3541 ataagaggtc agcaacgcac ccacggctcc tttcttcctg atccacgtcg tggccgctgt 3601 acgctctcaa acacgctttg gcgctgatgc gtgcctttat acaaagaaca aagaagaagc 3661 gaaaaacggg agcgtgttgc tttggggatg tatgcgtgcc acagttctgc ggtacaattc 3721 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa 3781 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac 3841 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca 3901 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc 3961 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc 4021 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc 4081 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag 4141 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc 4201 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt 4261 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct 4321 ttctcaatgc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg 4381 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct 4441 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat 4501 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg 4561 ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa 4621 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt 4681 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc 4741 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt 4801 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta 4861 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat 4921 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac 4981 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg 5041 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag 5101 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt 5161 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt 5221 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt 5281 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt 5341 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct 5401 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt 5461 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac 5521 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa 5581 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa 5641 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca 5701 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct 5761 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga 5821 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc 5881 tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag 5941 gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc 6001 ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc 6061 gtcagcgggt gttggcgggt gtcggggctg gcttaactat gcggcatcag agcagattgt 6121 actgagagtg cacgatatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg 6181 catcaggcgc cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc 6241 ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt 6301 aacgccaggg ttttcccagt cacgacgttg taaaacgacg gccagtgcca // LOCUS pPLOT-10TY-Puro-Puro 6850 bp ds-DNA circular 24-AUG-2016 DEFINITION pPLOT-10TY-Puro-Puro FEATURES Location/Qualifiers primer_bind 1..3 /label="T7" /ApEinfo_revcolor=#d59687 /ApEinfo_fwdcolor=#d59687 primer_bind 6826..6850 /label="T7" /ApEinfo_revcolor=#b7e6d7 /ApEinfo_fwdcolor=#b7e6d7 rep_origin complement(6347..6653) /label="F1 ori" /ApEinfo_revcolor=#75c6a9 /ApEinfo_fwdcolor=#75c6a9 misc_feature 1870..1899 /label="Ty8" /ApEinfo_revcolor=#c7b0e3 /ApEinfo_fwdcolor=#c7b0e3 primer_bind 6810..6827 /label="M13-fwd" /ApEinfo_revcolor=#ffef86 /ApEinfo_fwdcolor=#ffef86 rep_origin complement(4395..5077) /label="ColE1 origin" /ApEinfo_revcolor=#ff9ccd /ApEinfo_fwdcolor=#ff9ccd misc_feature 3183..3782 /label="PuroR" /ApEinfo_revcolor=#d59687 /ApEinfo_fwdcolor=#d59687 misc_feature 1654..1683 /label="Ty2" /ApEinfo_revcolor=#d6b295 /ApEinfo_fwdcolor=#d6b295 misc_feature 1618..1647 /label="Ty1" /ApEinfo_revcolor=#f8d3a9 /ApEinfo_fwdcolor=#f8d3a9 misc_feature 1582..1611 /label="GS linker" /ApEinfo_revcolor=#84b0dc /ApEinfo_fwdcolor=#84b0dc 3'UTR 3789..3937 /label="Ald3'UTR (short)(1)" /ApEinfo_revcolor=#c7b0e3 /ApEinfo_fwdcolor=#c7b0e3 misc_feature 1798..1827 /label="Ty6" /ApEinfo_revcolor=#c7b0e3 /ApEinfo_fwdcolor=#c7b0e3 misc_feature 1978..2007 /label="GS linker" /ApEinfo_revcolor=#d59687 /ApEinfo_fwdcolor=#d59687 misc_feature 1834..1863 /label="Ty7" /ApEinfo_revcolor=#b4abac /ApEinfo_fwdcolor=#b4abac misc_binding complement(4033..4055) /label="LacO" /ApEinfo_revcolor=#f8d3a9 /ApEinfo_fwdcolor=#f8d3a9 primer_bind complement(3976..4001) /label="SP6" /ApEinfo_revcolor=#84b0dc /ApEinfo_fwdcolor=#84b0dc misc_feature complement(1988..2007) /label="Primer N-terminal Rev" /ApEinfo_revcolor=#9eafd2 /ApEinfo_fwdcolor=#9eafd2 misc_feature 932..1488 /label="PGKB 5'" /ApEinfo_revcolor=#faac61 /ApEinfo_fwdcolor=#faac61 misc_feature 1906..1935 /label="Ty9" /ApEinfo_revcolor=#c6c9d1 /ApEinfo_fwdcolor=#c6c9d1 primer_bind complement(4007..4027) /label="M13-rev" /ApEinfo_revcolor=#b7e6d7 /ApEinfo_fwdcolor=#b7e6d7 primer_bind 6826..3 /label="T7" /ApEinfo_revcolor=#85dae9 /ApEinfo_fwdcolor=#85dae9 misc_feature 1582..1601 /label="Primer C-terminal For" /ApEinfo_revcolor=#ff9ccd /ApEinfo_fwdcolor=#ff9ccd primer_bind complement(3938..3957) /label="#162" /ApEinfo_revcolor=#f58a5e /ApEinfo_fwdcolor=#f58a5e misc_feature 777..925 /label="Ald3'UTR" /ApEinfo_revcolor=#b1ff67 /ApEinfo_fwdcolor=#b1ff67 CDS complement(6671..6739) /label="LacZ alpha" /ApEinfo_revcolor=#b4abac /ApEinfo_fwdcolor=#b4abac misc_feature 171..770 /label="PuroR" /ApEinfo_revcolor=#d59687 /ApEinfo_fwdcolor=#d59687 misc_feature 2101..3176 /label="Cf PGKA/B" /ApEinfo_revcolor=#c7b0e3 /ApEinfo_fwdcolor=#c7b0e3 misc_feature 1942..1971 /label="Ty10" /ApEinfo_revcolor=#f58a5e /ApEinfo_fwdcolor=#f58a5e misc_feature 1762..1791 /label="Ty5" /ApEinfo_revcolor=#9eafd2 /ApEinfo_fwdcolor=#9eafd2 CDS complement(5175..5834) /label="AmpR" /ApEinfo_revcolor=#d6b295 /ApEinfo_fwdcolor=#d6b295 CDS 2008..2097 /label="3xMyc" /ApEinfo_revcolor=#ffef86 /ApEinfo_fwdcolor=#ffef86 misc_feature 1690..1719 /label="Ty3" /ApEinfo_revcolor=#ff9ccd /ApEinfo_fwdcolor=#ff9ccd 5'UTR 36..164 /label="Actin 5'" /ApEinfo_revcolor=#b7e6d7 /ApEinfo_fwdcolor=#b7e6d7 primer_bind 16..35 /label="#161" /ApEinfo_revcolor=#f58a5e /ApEinfo_fwdcolor=#f58a5e CDS 1492..1581 /label="3xMyc" /ApEinfo_revcolor=#ffef86 /ApEinfo_fwdcolor=#ffef86 misc_feature 1726..1755 /label="Ty4" /ApEinfo_revcolor=#ff9ccd /ApEinfo_fwdcolor=#ff9ccd ORIGIN 1 GGGCGAATTG GGCCCgtata atgcagacct gctgcAAGCT AGGGGCACAG CAAGGTCTTC 61 TGAAATTCAT GTTTTTTTTT TTTTTACTCT GCATTGCAGT CTCCGCTCTT ATTTAGTTTT 121 GCTTTACGTA AGGTCTCGTT GCTGCCATAA AATAAGCTCT AGTGgaattc ATGACTGAAT 181 ACAAGCCAAC GGTTCGTCTT GCTACGCGAG ACGATGTGCC GCGGGCCGTA CGGACGCTGG 241 CTGCTGCGTT CGCTGATTAT CCCGCCACCC GACATACGGT TGACCCAGAT CGACACATTG 301 AGCGAGTCAC AGAACTTCAA GAGTTATTCT TAACACGGGT GGGCTTGGAC ATTGGTAAGG 361 TTTGGGTTGC GGACGATGGC GCGGCTGTTG CGGTGTGGAC GACGCCAGAG TCAGTCGAAG 421 CGGGCGCAGT GTTTGCTGAG ATCGGTCCCC GAATGGCGGA GCTTAGCGGG TCACGCCTCG 481 CTGCACAACA GCAGATGGAA GGTCTGTTGG CACCACATAG ACCTAAGGAG CCGGCTTGGT 541 TCTTAGCGAC AGTTGGCGTC AGTCCGGACC ACCAAGGTAA GGGGCTGGGT TCAGCGGTTG 601 TTCTTCCAGG AGTAGAGGCT GCGGAAAGGG CAGGTGTGCC GGCGTTCCTT GAAACGTCTG 661 CACCACGTAA CCTTCCTTTT TACGAGAGAT TGGGGTTTAC AGTAACAGCA GATGTTGAAG 721 TGCCTGAAGG GCCGAGGACA TGGTGCATGA CACGTAAGCC GGGAGCCTAA ccatggtgcc 781 catttagttg gcttttccct tgtctcgtgt cttttccgtg gaaaggttcc cggagtaatc 841 tgatggcaca gcagggaggt gcgcctgcag gttggttagg aaggggggat gatgtaaaag 901 aagaaaatgg ggggattcga gccccacgcg ttcgagaccg acaagaccag aatagcgctt 961 atgtgtgtgt gtttgtgtgt gggtgtgtgt gcccatgtgt gacaaagagg ccagtgcttt 1021 ctctaaagca agagcgcgag gttgctggct aaggactcta agtccctcac agtgatggcg 1081 tgttgcattt tctaaaccgt caatccaaca cgtcggtagc atatcggttt tgtaggctct 1141 tctggatacc ttccccagta agaccgccac tgcttcgtga ctgaagcact gtaaatatca 1201 ctgaaaccgt cgcgttgctt tgtctaatta tcaccgcctt tgccagctat cgcctcaggc 1261 tgcgctcttt gactaatcca ctcaccactt ctctgccttc ttcctccggt tgttgttgtg 1321 tcctgcgtgt acatggcgcg tgtccttttc gagcaaacag ctgtcttttc tttcacgata 1381 acacactcat attaaacgcg agtattactc atcagtcaac gtcacattcc gctctgtcca 1441 cttcgacctt acacctctac ttgtccaact atcttccact tgtcaagcat ggaacagaag 1501 ctgatcagcg aagaagacct ggagcaaaag ctcattagcg aggaggacct cgagcagaag 1561 ctgattagcg aggaggacct gggttctggt agtggttccg gttccggttc taagcttGAG 1621 GTACACACAA ACCAGGACCC ACTGGACGGC TCCGAGGTGC ATACCAATCA AGATCCGCTG 1681 GACGGCTCTG AAGTTCACAC CAATCAAGAT CCCTTGGATG GCTCCGAAGT GCACACTAAT 1741 CAGGACCCTC TGGACGGGAG TGAGGTGCAC ACGAACCAAG ATCCACTGGA CGGTAGTGAA 1801 GTTCATACCA ACCAGGACCC ATTGGACGGT TCGGAGGTTC ATACGAATCA AGACCCCTTA 1861 GACGGTTCTG AGGTACATAC AAATCAGGAC CCCCTCGATG GTAGTGAAGT CCATACAAAT 1921 CAGGACCCGC TGGATGGCTC AGAGGTTCAT ACGAACCAAG ATCCTCTTGA Tggatccgga 1981 tcaggatctg gatcaggatc gggtagtgaa cagaagctga tcagcgaaga agacctggag 2041 caaaagctca ttagcgagga ggacctcgag cagaagctga ttagcgagga ggacctgtaa 2101 ccaccaccac cactgagaat tctgtattac gccgttttaa gagctccttc gacgcctttt 2161 tgtccttcag ctgctccacg gtgactcgct tctctctctt tccacagtgt ctcttttttc 2221 ttttcactct ctatacaaat gtgagcgacc tccttttctg tacaacggcc ttccggcgtg 2281 tgcttttctc atcgacctct cctcgcttct tgggcactcc ttcatcgagc aaacaagacg 2341 agggaggtga cgcgattggt gagaccacta cgagtatgga cggagctgtc tgataccccc 2401 tgttttttat cttgtacccc gctctgcagt gacggtagcc cgctgctgtg cttcggtatc 2461 gccgcttcat acgcttctct cttttttcaa cgtgcggcgc tgattcaaag gttacctcaa 2521 cgcgacccgc cgctgcatcc tttgttgctc ctcttgtcgc aaacaaacaa aaaacgtgct 2581 gtgctttttc ctttatcgtg tctttttgca aagtctagag tttaccgaca agaccagaat 2641 agcgcttatg tgtgtgtgtt tgtgtgtggg tgtgtgtgcc catgtgtgac aaagaggcca 2701 gtgctttctc taaagcaaga gcgcgaggtt gctggctaag gactctaagt ccctcacagt 2761 gatggcgtgt tgcattttct aaaccgtcaa tccaacacgt cggtagcata tcggttttgt 2821 aggctcttct ggataccttc cccagtaaga ccgccactgc ttcgtgactg aagcactgta 2881 aatatcactg aaaccgtcgc gttgctttgt ctaattatca ccgcctttgc cagctatcgc 2941 ctcaggctgc gctctttgac taatccactc accacttctc tgccttcttc ctccggttgt 3001 tgttgtgtcc tgcgtgtaca tggcgcgtgt ccttttcgag caaacagctg tcttttcttt 3061 cacgataaca cactcatatt aaacgcgagt attactcatc agtcaacgtc acattccgct 3121 ctgtccactt cgaccttaca cctctacttg tccaactatc ttccacttgt caagcggcta 3181 gcATGACTGA ATACAAGCCA ACGGTTCGTC TTGCTACGCG AGACGATGTG CCGCGGGCCG 3241 TACGGACGCT GGCTGCTGCG TTCGCTGATT ATCCCGCCAC CCGACATACG GTTGACCCAG 3301 ATCGACACAT TGAGCGAGTC ACAGAACTTC AAGAGTTATT CTTAACACGG GTGGGCTTGG 3361 ACATTGGTAA GGTTTGGGTT GCGGACGATG GCGCGGCTGT TGCGGTGTGG ACGACGCCAG 3421 AGTCAGTCGA AGCGGGCGCA GTGTTTGCTG AGATCGGTCC CCGAATGGCG GAGCTTAGCG 3481 GGTCACGCCT CGCTGCACAA CAGCAGATGG AAGGTCTGTT GGCACCACAT AGACCTAAGG 3541 AGCCGGCTTG GTTCTTAGCG ACAGTTGGCG TCAGTCCGGA CCACCAAGGT AAGGGGCTGG 3601 GTTCAGCGGT TGTTCTTCCA GGAGTAGAGG CTGCGGAAAG GGCAGGTGTG CCGGCGTTCC 3661 TTGAAACGTC TGCACCACGT AACCTTCCTT TTTACGAGAG ATTGGGGTTT ACAGTAACAG 3721 CAGATGTTGA AGTGCCTGAA GGGCCGAGGA CATGGTGCAT GACACGTAAG CCGGGAGCCT 3781 AActcgagTG CCCATTTAGT TGGCTTTTCC CTTGTCTCGT GTCTTTTCCG TGGAAAGGTT 3841 CCCGGAGTAA TCTGATGGCA CAGCAGGGAG GTGCGCCTGC AGGTTGGTTA GGAAGGGGGG 3901 ATGATGTAAA AGAAGAAAAT GGGGGGATTC GAGCCCCGCA CAGGTCTCTC AAATTGGATG 3961 CATAGCTTGA GTATTCTATA GTGTCACCTA AATAGCTTGG CGTAATCATG GTCATAGCTG 4021 TTTCCTGTGT GAAATTGTTA TCCGCTCACA ATTCCACACA ACATACGAGC CGGAAGCATA 4081 AAGTGTAAAG CCTGGGGTGC CTAATGAGTG AGCTAACTCA CATTAATTGC GTTGCGCTCA 4141 CTGCCCGCTT TCCAGTCGGG AAACCTGTCG TGCCAGCTGC ATTAATGAAT CGGCCAACGC 4201 GCGGGGAGAG GCGGTTTGCG TATTGGGCGC TCTTCCGCTT CCTCGCTCAC TGACTCGCTG 4261 CGCTCGGTCG TTCGGCTGCG GCGAGCGGTA TCAGCTCACT CAAAGGCGGT AATACGGTTA 4321 TCCACAGAAT CAGGGGATAA CGCAGGAAAG AACATGTGAG CAAAAGGCCA GCAAAAGGCC 4381 AGGAACCGTA AAAAGGCCGC GTTGCTGGCG TTTTTCCATA GGCTCCGCCC CCCTGACGAG 4441 CATCACAAAA ATCGACGCTC AAGTCAGAGG TGGCGAAACC CGACAGGACT ATAAAGATAC 4501 CAGGCGTTTC CCCCTGGAAG CTCCCTCGTG CGCTCTCCTG TTCCGACCCT GCCGCTTACC 4561 GGATACCTGT CCGCCTTTCT CCCTTCGGGA AGCGTGGCGC TTTCTCATAG CTCACGCTGT 4621 AGGTATCTCA GTTCGGTGTA GGTCGTTCGC TCCAAGCTGG GCTGTGTGCA CGAACCCCCC 4681 GTTCAGCCCG ACCGCTGCGC CTTATCCGGT AACTATCGTC TTGAGTCCAA CCCGGTAAGA 4741 CACGACTTAT CGCCACTGGC AGCAGCCACT GGTAACAGGA TTAGCAGAGC GAGGTATGTA 4801 GGCGGTGCTA CAGAGTTCTT GAAGTGGTGG CCTAACTACG GCTACACTAG AAGAACAGTA 4861 TTTGGTATCT GCGCTCTGCT GAAGCCAGTT ACCTTCGGAA AAAGAGTTGG TAGCTCTTGA 4921 TCCGGCAAAC AAACCACCGC TGGTAGCGGT GGTTTTTTTG TTTGCAAGCA GCAGATTACG 4981 CGCAGAAAAA AAGGATCTCA AGAAGATCCT TTGATCTTTT CTACGGGGTC TGACGCTCAG 5041 TGGAACGAAA ACTCACGTTA AGGGATTTTG GTCATGAGAT TATCAAAAAG GATCTTCACC 5101 TAGATCCTTT TAAATTAAAA ATGAAGTTTT AAATCAATCT AAAGTATATA TGAGTAAACT 5161 TGGTCTGACA GTTACCAATG CTTAATCAGT GAGGCACCTA TCTCAGCGAT CTGTCTATTT 5221 CGTTCATCCA TAGTTGCCTG ACTCCCCGTC GTGTAGATAA CTACGATACG GGAGGGCTTA 5281 CCATCTGGCC CCAGTGCTGC AATGATACCG CGAGACCCAC GCTCACCGGC TCCAGATTTA 5341 TCAGCAATAA ACCAGCCAGC CGGAAGGGCC GAGCGCAGAA GTGGTCCTGC AACTTTATCC 5401 GCCTCCATCC AGTCTATTAA TTGTTGCCGG GAAGCTAGAG TAAGTAGTTC GCCAGTTAAT 5461 AGTTTGCGCA ACGTTGTTGC CATTGCTACA GGCATCGTGG TGTCACGCTC GTCGTTTGGT 5521 ATGGCTTCAT TCAGCTCCGG TTCCCAACGA TCAAGGCGAG TTACATGATC CCCCATGTTG 5581 TGCAAAAAAG CGGTTAGCTC CTTCGGTCCT CCGATCGTTG TCAGAAGTAA GTTGGCCGCA 5641 GTGTTATCAC TCATGGTTAT GGCAGCACTG CATAATTCTC TTACTGTCAT GCCATCCGTA 5701 AGATGCTTTT CTGTGACTGG TGAGTACTCA ACCAAGTCAT TCTGAGAATA GTGTATGCGG 5761 CGACCGAGTT GCTCTTGCCC GGCGTCAATA CGGGATAATA CCGCGCCACA TAGCAGAACT 5821 TTAAAAGTGC TCATCATTGG AAAACGTTCT TCGGGGCGAA AACTCTCAAG GATCTTACCG 5881 CTGTTGAGAT CCAGTTCGAT GTAACCCACT CGTGCACCCA ACTGATCTTC AGCATCTTTT 5941 ACTTTCACCA GCGTTTCTGG GTGAGCAAAA ACAGGAAGGC AAAATGCCGC AAAAAAGGGA 6001 ATAAGGGCGA CACGGAAATG TTGAATACTC ATACTCTTCC TTTTTCAATA TTATTGAAGC 6061 ATTTATCAGG GTTATTGTCT CATGAGCGGA TACATATTTG AATGTATTTA GAAAAATAAA 6121 CAAATAGGGG TTCCGCGCAC ATTTCCCCGA AAAGTGCCAC CTGATGCGGT GTGAAATACC 6181 GCACAGATGC GTAAGGAGAA AATACCGCAT CAGGAAATTG TAAGCGTTAA TATTTTGTTA 6241 AAATTCGCGT TAAATTTTTG TTAAATCAGC TCATTTTTTA ACCAATAGGC CGAAATCGGC 6301 AAAATCCCTT ATAAATCAAA AGAATAGACC GAGATAGGGT TGAGTGTTGT TCCAGTTTGG 6361 AACAAGAGTC CACTATTAAA GAACGTGGAC TCCAACGTCA AAGGGCGAAA AACCGTCTAT 6421 CAGGGCGATG GCCCACTACG TGAACCATCA CCCTAATCAA GTTTTTTGGG GTCGAGGTGC 6481 CGTAAAGCAC TAAATCGGAA CCCTAAAGGG AGCCCCCGAT TTAGAGCTTG ACGGGGAAAG 6541 CCGGCGAACG TGGCGAGAAA GGAAGGGAAG AAAGCGAAAG GAGCGGGCGC TAGGGCGCTG 6601 GCAAGTGTAG CGGTCACGCT GCGCGTAACC ACCACACCCG CCGCGCTTAA TGCGCCGCTA 6661 CAGGGCGCGT CCATTCGCCA TTCAGGCTGC GCAACTGTTG GGAAGGGCGA TCGGTGCGGG 6721 CCTCTTCGCT ATTACGCCAG CTGGCGAAAG GGGGATGTGC TGCAAGGCGA TTAAGTTGGG 6781 TAACGCCAGG GTTTTCCCAG TCACGACGTT GTAAAACGAC GGCCAGTGAA TTGTAATACG 6841 ACTCACTATA // LOCUS pPLOT-BirA-Puro-Puro 7465 bp ds-DNA circular SYN 30-AUG-2016 DEFINITION pPLOT-BirA-Puro-Puro ACCESSION . VERSION . KEYWORDS pPLOT-BirA-puro SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7465) AUTHORS . TITLE Direct Submission JOURNAL Exported Tuesday, Aug 30, 2016 from SnapGene Viewer 3.1.2 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7465 /organism="synthetic DNA construct" /mol_type="other DNA" misc_feature 16..35 /note="#161" 5'UTR 36..164 /note="Actin 5'" CDS 171..770 /codon_start=1 /note="PuroR" /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA" 3'UTR 777..925 /note="Ald3'UTR" 5'UTR 932..1488 /note="PGKB 5'" CDS 1492..1581 /codon_start=1 /product="3 tandem Myc epitope tags" /note="3xMyc" /translation="EQKLISEEDLEQKLISEEDLEQKLISEEDL" misc_feature 1582..1611 /note="GS linker" misc_feature 1618..2586 /note="BirA*" misc_feature 2593..2622 /note="GS linker" CDS 2623..2712 /codon_start=1 /product="3 tandem Myc epitope tags" /note="3xMyc" /translation="EQKLISEEDLEQKLISEEDLEQKLISEEDL" misc_feature 2716..3791 /note="Cf PGKA/B" CDS 3798..4397 /codon_start=1 /note="PuroR" /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA" 3'UTR 4404..4552 /note="Ald3'UTR" misc_feature complement(4553..4572) /note="#162" promoter complement(4590..4608) /note="SP6 promoter" /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind complement(4626..4642) /note="M13 rev" /note="common sequencing primer, one of multiple similar variants" protein_bind 4650..4666 /bound_moiety="lac repressor encoded by lacI" /note="lac operator" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." promoter complement(4674..4704) /note="lac promoter" /note="promoter for the E. coli lac operon" protein_bind 4719..4740 /bound_moiety="E. coli catabolite activator protein" /note="CAP binding site" /note="CAP binding activates transcription in the presence of cAMP." rep_origin complement(5028..5616) /direction=LEFT /note="ori" /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(5787..6647) /codon_start=1 /gene="bla" /product="beta-lactamase" /note="AmpR" /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" promoter complement(6648..6752) /gene="bla" /note="AmpR promoter" rep_origin complement(6830..7285) /direction=LEFT /note="f1 ori" /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind 7426..7442 /note="M13 fwd" /note="common sequencing primer, one of multiple similar variants" promoter 7449..2 /note="T7 promoter" /note="promoter for bacteriophage T7 RNA polymerase" ORIGIN 1 gggcgaattg ggcccgtata atgcagacct gctgcaagct aggggcacag caaggtcttc 61 tgaaattcat gttttttttt tttttactct gcattgcagt ctccgctctt atttagtttt 121 gctttacgta aggtctcgtt gctgccataa aataagctct agtggaattc atgactgaat 181 acaagccaac ggttcgtctt gctacgcgag acgatgtgcc gcgggccgta cggacgctgg 241 ctgctgcgtt cgctgattat cccgccaccc gacatacggt tgacccagat cgacacattg 301 agcgagtcac agaacttcaa gagttattct taacacgggt gggcttggac attggtaagg 361 tttgggttgc ggacgatggc gcggctgttg cggtgtggac gacgccagag tcagtcgaag 421 cgggcgcagt gtttgctgag atcggtcccc gaatggcgga gcttagcggg tcacgcctcg 481 ctgcacaaca gcagatggaa ggtctgttgg caccacatag acctaaggag ccggcttggt 541 tcttagcgac agttggcgtc agtccggacc accaaggtaa ggggctgggt tcagcggttg 601 ttcttccagg agtagaggct gcggaaaggg caggtgtgcc ggcgttcctt gaaacgtctg 661 caccacgtaa ccttcctttt tacgagagat tggggtttac agtaacagca gatgttgaag 721 tgcctgaagg gccgaggaca tggtgcatga cacgtaagcc gggagcctaa ccatggtgcc 781 catttagttg gcttttccct tgtctcgtgt cttttccgtg gaaaggttcc cggagtaatc 841 tgatggcaca gcagggaggt gcgcctgcag gttggttagg aaggggggat gatgtaaaag 901 aagaaaatgg ggggattcga gccccacgcg ttcgagaccg acaagaccag aatagcgctt 961 atgtgtgtgt gtttgtgtgt gggtgtgtgt gcccatgtgt gacaaagagg ccagtgcttt 1021 ctctaaagca agagcgcgag gttgctggct aaggactcta agtccctcac agtgatggcg 1081 tgttgcattt tctaaaccgt caatccaaca cgtcggtagc atatcggttt tgtaggctct 1141 tctggatacc ttccccagta agaccgccac tgcttcgtga ctgaagcact gtaaatatca 1201 ctgaaaccgt cgcgttgctt tgtctaatta tcaccgcctt tgccagctat cgcctcaggc 1261 tgcgctcttt gactaatcca ctcaccactt ctctgccttc ttcctccggt tgttgttgtg 1321 tcctgcgtgt acatggcgcg tgtccttttc gagcaaacag ctgtcttttc tttcacgata 1381 acacactcat attaaacgcg agtattactc atcagtcaac gtcacattcc gctctgtcca 1441 cttcgacctt acacctctac ttgtccaact atcttccact tgtcaagcat ggaacagaag 1501 ctgatcagcg aagaagacct ggagcaaaag ctcattagcg aggaggacct cgagcagaag 1561 ctgattagcg aggaggacct gggttctggt agtggttccg gttccggttc taagcttgac 1621 aaggacaaca ccgtgcccct gaagctgatc gccctgctgg ccaacggcga gttccactct 1681 ggcgagcagc tgggagagac cctgggaatg agcagagccg ccatcaacaa gcacatccag 1741 acactgagag actggggagt ggacgtgttc accgtgcctg gcaagggcta cagcctgcct 1801 gagcctatcc agctgctgaa cgccaagcag atcctgggac agctggatgg cggaagcgtg 1861 gccgtgctgc ctgtgatcga ctccaccaat cagtacctgc tggacagaat cggagagctg 1921 aagtccggcg acgcctgcat cgccgagtac cagcaggctg gcagaggagg cagaggacgg 1981 aagtggttca gcccattcgg agccaacctg tacctgtcca tgttctggag actggagcag 2041 ggacctgctg ctgccatcgg actgagtctg gtgatcggaa tcgtgatggc cgaggtgctg 2101 agaaagctgg gagccgacaa ggtgagagtg aagtggccta atgacctgta cctccaggac 2161 cgcaagctgg ctggcatcct ggtggagctg acaggcaaga caggcgatgc cgctcagatc 2221 gtgatcggag ccggaatcaa catggccatg agaagagtgg aggagagcgt ggtgaaccag 2281 ggctggatca ccctgcagga ggctggcatc aacctggacc ggaacaccct ggccgccatg 2341 ctgatcagag agctgagagc cgctctggag ctgttcgagc aggagggact ggctccttac 2401 ctgagcagat gggagaagct ggacaacttc atcaacagac ctgtgaagct gatcatcggc 2461 gacaaggaaa tcttcggcat ctccagagga atcgacaagc agggagctct gctgctggag 2521 caggacggaa tcatcaagcc ctggatgggc ggagaaatct ccctgagaag cgcagagaag 2581 ctcgagggat ccggatcagg atctggatca ggatcgggta gtgaacagaa gctgatcagc 2641 gaagaagacc tggagcaaaa gctcattagc gaggaggacc tcgagcagaa gctgattagc 2701 gaggaggacc tgtaaccacc accaccactg agaattctgt attacgccgt tttaagagct 2761 ccttcgacgc ctttttgtcc ttcagctgct ccacggtgac tcgcttctct ctctttccac 2821 agtgtctctt ttttcttttc actctctata caaatgtgag cgacctcctt ttctgtacaa 2881 cggccttccg gcgtgtgctt ttctcatcga cctctcctcg cttcttgggc actccttcat 2941 cgagcaaaca agacgaggga ggtgacgcga ttggtgagac cactacgagt atggacggag 3001 ctgtctgata ccccctgttt tttatcttgt accccgctct gcagtgacgg tagcccgctg 3061 ctgtgcttcg gtatcgccgc ttcatacgct tctctctttt ttcaacgtgc ggcgctgatt 3121 caaaggttac ctcaacgcga cccgccgctg catcctttgt tgctcctctt gtcgcaaaca 3181 aacaaaaaac gtgctgtgct ttttccttta tcgtgtcttt ttgcaaagtc tagagtttac 3241 cgacaagacc agaatagcgc ttatgtgtgt gtgtttgtgt gtgggtgtgt gtgcccatgt 3301 gtgacaaaga ggccagtgct ttctctaaag caagagcgcg aggttgctgg ctaaggactc 3361 taagtccctc acagtgatgg cgtgttgcat tttctaaacc gtcaatccaa cacgtcggta 3421 gcatatcggt tttgtaggct cttctggata ccttccccag taagaccgcc actgcttcgt 3481 gactgaagca ctgtaaatat cactgaaacc gtcgcgttgc tttgtctaat tatcaccgcc 3541 tttgccagct atcgcctcag gctgcgctct ttgactaatc cactcaccac ttctctgcct 3601 tcttcctccg gttgttgttg tgtcctgcgt gtacatggcg cgtgtccttt tcgagcaaac 3661 agctgtcttt tctttcacga taacacactc atattaaacg cgagtattac tcatcagtca 3721 acgtcacatt ccgctctgtc cacttcgacc ttacacctct acttgtccaa ctatcttcca 3781 cttgtcaagc ggctagcatg actgaataca agccaacggt tcgtcttgct acgcgagacg 3841 atgtgccgcg ggccgtacgg acgctggctg ctgcgttcgc tgattatccc gccacccgac 3901 atacggttga cccagatcga cacattgagc gagtcacaga acttcaagag ttattcttaa 3961 cacgggtggg cttggacatt ggtaaggttt gggttgcgga cgatggcgcg gctgttgcgg 4021 tgtggacgac gccagagtca gtcgaagcgg gcgcagtgtt tgctgagatc ggtccccgaa 4081 tggcggagct tagcgggtca cgcctcgctg cacaacagca gatggaaggt ctgttggcac 4141 cacatagacc taaggagccg gcttggttct tagcgacagt tggcgtcagt ccggaccacc 4201 aaggtaaggg gctgggttca gcggttgttc ttccaggagt agaggctgcg gaaagggcag 4261 gtgtgccggc gttccttgaa acgtctgcac cacgtaacct tcctttttac gagagattgg 4321 ggtttacagt aacagcagat gttgaagtgc ctgaagggcc gaggacatgg tgcatgacac 4381 gtaagccggg agcctaactc gagtgcccat ttagttggct tttcccttgt ctcgtgtctt 4441 ttccgtggaa aggttcccgg agtaatctga tggcacagca gggaggtgcg cctgcaggtt 4501 ggttaggaag gggggatgat gtaaaagaag aaaatggggg gattcgagcc ccgcacaggt 4561 ctctcaaatt ggatgcatag cttgagtatt ctatagtgtc acctaaatag cttggcgtaa 4621 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata 4681 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta 4741 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa 4801 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg 4861 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag 4921 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa 4981 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc 5041 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca 5101 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg 5161 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct 5221 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt 5281 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag 5341 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc 5401 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac 5461 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga 5521 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc 5581 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg 5641 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca 5701 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt 5761 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca 5821 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg 5881 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca 5941 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt 6001 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt 6061 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca 6121 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca 6181 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga 6241 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact 6301 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga 6361 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg 6421 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc 6481 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga 6541 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat 6601 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt 6661 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt 6721 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgat 6781 gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcagga aattgtaagc 6841 gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa 6901 taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt 6961 gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg 7021 cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt 7081 ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga 7141 gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg 7201 ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg 7261 cttaatgcgc cgctacaggg cgcgtccatt cgccattcag gctgcgcaac tgttgggaag 7321 ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa 7381 ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca 7441 gtgaattgta atacgactca ctata // LOCUS pPLOT-TY::Halo::TY-Puro 7465 bp ds-DNA circular 24-AUG-2016 DEFINITION pPLOT-Halo-Puro-Puro FEATURES Location/Qualifiers misc_feature 1651..2556 /label="HaloTag (promega)" /ApEinfo_revcolor=#75c6a9 /ApEinfo_fwdcolor=#75c6a9 misc_feature 3798..4397 /label="PuroR" /ApEinfo_revcolor=#d59687 /ApEinfo_fwdcolor=#d59687 misc_feature 1582..1611 /label="GS linker" /ApEinfo_revcolor=#84b0dc /ApEinfo_fwdcolor=#84b0dc CDS complement(7286..7354) /label="LacZ alpha" /ApEinfo_revcolor=#b4abac /ApEinfo_fwdcolor=#b4abac primer_bind complement(4553..4572) /label="#162" /ApEinfo_revcolor=#f58a5e /ApEinfo_fwdcolor=#f58a5e primer_bind complement(4591..4616) /label="SP6" /ApEinfo_revcolor=#84b0dc /ApEinfo_fwdcolor=#84b0dc primer_bind 7441..3 /label="T7" /ApEinfo_revcolor=#85dae9 /ApEinfo_fwdcolor=#85dae9 misc_feature 777..925 /label="Ald3'UTR" /ApEinfo_revcolor=#b1ff67 /ApEinfo_fwdcolor=#b1ff67 rep_origin complement(5010..5692) /label="ColE1 origin" /ApEinfo_revcolor=#ff9ccd /ApEinfo_fwdcolor=#ff9ccd CDS 1492..1581 /label="3xMyc" /ApEinfo_revcolor=#ffef86 /ApEinfo_fwdcolor=#ffef86 primer_bind complement(4622..4642) /label="M13-rev" /ApEinfo_revcolor=#b7e6d7 /ApEinfo_fwdcolor=#b7e6d7 rep_origin complement(6962..7268) /label="F1 ori" /ApEinfo_revcolor=#75c6a9 /ApEinfo_fwdcolor=#75c6a9 primer_bind 7425..7442 /label="M13-fwd" /ApEinfo_revcolor=#ffef86 /ApEinfo_fwdcolor=#ffef86 CDS complement(5790..6449) /label="AmpR" /ApEinfo_revcolor=#d6b295 /ApEinfo_fwdcolor=#d6b295 misc_feature 2593..2622 /label="GS linker" /ApEinfo_revcolor=#d59687 /ApEinfo_fwdcolor=#d59687 misc_feature 1618..1650 /label="Ty" /ApEinfo_revcolor=#faac61 /ApEinfo_fwdcolor=#faac61 misc_feature 2716..3791 /label="Cf PGKA/B" /ApEinfo_revcolor=#c7b0e3 /ApEinfo_fwdcolor=#c7b0e3 misc_binding complement(4648..4670) /label="LacO" /ApEinfo_revcolor=#f8d3a9 /ApEinfo_fwdcolor=#f8d3a9 misc_feature 2557..2586 /label="Ty(1)" /ApEinfo_revcolor=#85dae9 /ApEinfo_fwdcolor=#85dae9 5'UTR 36..164 /label="Actin 5'" /ApEinfo_revcolor=#b7e6d7 /ApEinfo_fwdcolor=#b7e6d7 misc_feature 171..770 /label="PuroR" /ApEinfo_revcolor=#d59687 /ApEinfo_fwdcolor=#d59687 3'UTR 4404..4552 /label="Ald3'UTR (short)(1)" /ApEinfo_revcolor=#c7b0e3 /ApEinfo_fwdcolor=#c7b0e3 primer_bind 1..3 /label="T7" /ApEinfo_revcolor=#d59687 /ApEinfo_fwdcolor=#d59687 misc_feature complement(2603..2622) /label="Primer N-terminal Rev" /ApEinfo_revcolor=#9eafd2 /ApEinfo_fwdcolor=#9eafd2 primer_bind 16..35 /label="#161" /ApEinfo_revcolor=#f58a5e /ApEinfo_fwdcolor=#f58a5e primer_bind 7441..7465 /label="T7" /ApEinfo_revcolor=#b7e6d7 /ApEinfo_fwdcolor=#b7e6d7 misc_feature 1582..1601 /label="Primer C-terminal For" /ApEinfo_revcolor=#ff9ccd /ApEinfo_fwdcolor=#ff9ccd CDS 2623..2712 /label="3xMyc" /ApEinfo_revcolor=#ffef86 /ApEinfo_fwdcolor=#ffef86 misc_feature 932..1488 /label="PGKB 5'" /ApEinfo_revcolor=#faac61 /ApEinfo_fwdcolor=#faac61 ORIGIN 1 GGGCGAATTG GGCCCgtata atgcagacct gctgcAAGCT AGGGGCACAG CAAGGTCTTC 61 TGAAATTCAT GTTTTTTTTT TTTTTACTCT GCATTGCAGT CTCCGCTCTT ATTTAGTTTT 121 GCTTTACGTA AGGTCTCGTT GCTGCCATAA AATAAGCTCT AGTGgaattc ATGACTGAAT 181 ACAAGCCAAC GGTTCGTCTT GCTACGCGAG ACGATGTGCC GCGGGCCGTA CGGACGCTGG 241 CTGCTGCGTT CGCTGATTAT CCCGCCACCC GACATACGGT TGACCCAGAT CGACACATTG 301 AGCGAGTCAC AGAACTTCAA GAGTTATTCT TAACACGGGT GGGCTTGGAC ATTGGTAAGG 361 TTTGGGTTGC GGACGATGGC GCGGCTGTTG CGGTGTGGAC GACGCCAGAG TCAGTCGAAG 421 CGGGCGCAGT GTTTGCTGAG ATCGGTCCCC GAATGGCGGA GCTTAGCGGG TCACGCCTCG 481 CTGCACAACA GCAGATGGAA GGTCTGTTGG CACCACATAG ACCTAAGGAG CCGGCTTGGT 541 TCTTAGCGAC AGTTGGCGTC AGTCCGGACC ACCAAGGTAA GGGGCTGGGT TCAGCGGTTG 601 TTCTTCCAGG AGTAGAGGCT GCGGAAAGGG CAGGTGTGCC GGCGTTCCTT GAAACGTCTG 661 CACCACGTAA CCTTCCTTTT TACGAGAGAT TGGGGTTTAC AGTAACAGCA GATGTTGAAG 721 TGCCTGAAGG GCCGAGGACA TGGTGCATGA CACGTAAGCC GGGAGCCTAA ccatggtgcc 781 catttagttg gcttttccct tgtctcgtgt cttttccgtg gaaaggttcc cggagtaatc 841 tgatggcaca gcagggaggt gcgcctgcag gttggttagg aaggggggat gatgtaaaag 901 aagaaaatgg ggggattcga gccccacgcg ttcgagaccg acaagaccag aatagcgctt 961 atgtgtgtgt gtttgtgtgt gggtgtgtgt gcccatgtgt gacaaagagg ccagtgcttt 1021 ctctaaagca agagcgcgag gttgctggct aaggactcta agtccctcac agtgatggcg 1081 tgttgcattt tctaaaccgt caatccaaca cgtcggtagc atatcggttt tgtaggctct 1141 tctggatacc ttccccagta agaccgccac tgcttcgtga ctgaagcact gtaaatatca 1201 ctgaaaccgt cgcgttgctt tgtctaatta tcaccgcctt tgccagctat cgcctcaggc 1261 tgcgctcttt gactaatcca ctcaccactt ctctgccttc ttcctccggt tgttgttgtg 1321 tcctgcgtgt acatggcgcg tgtccttttc gagcaaacag ctgtcttttc tttcacgata 1381 acacactcat attaaacgcg agtattactc atcagtcaac gtcacattcc gctctgtcca 1441 cttcgacctt acacctctac ttgtccaact atcttccact tgtcaagcat ggaacagaag 1501 ctgatcagcg aagaagacct ggagcaaaag ctcattagcg aggaggacct cgagcagaag 1561 ctgattagcg aggaggacct gggttctggt agtggttccg gttccggttc taagcttATG 1621 GAGGTTCTCA CTAACCAAGA TCCACTTGAT ACTAGTGGTA GTGCAGAAAT CGGTACTGGC 1681 TTTCCATTCG ACCCCCATTA TGTGGAAGTC CTGGGCGAGC GCATGCACTA CGTCGATGTT 1741 GGTCCGCGCG ATGGCACCCC TGTGCTGTTC CTGCACGGTA ACCCGACCTC CTCCTACGTG 1801 TGGCGCAACA TCATCCCGCA TGTTGCACCG ACCCATCGCT GCATTGCTCC AGACCTGATC 1861 GGTATGGGCA AATCCGACAA ACCAGACCTG GGTTATTTCT TCGACGACCA CGTCCGCTTC 1921 ATGGATGCCT TCATCGAAGC CCTGGGTCTG GAAGAGGTCG TCCTGGTCAT TCACGACTGG 1981 GGCTCCGCTC TGGGTTTCCA CTGGGCCAAG CGCAATCCAG AGCGCGTCAA AGGTATTGCA 2041 TTTATGGAGT TCATCCGCCC TATCCCGACC TGGGACGAAT GGCCAGAATT TGCCCGCGAG 2101 ACCTTCCAGG CCTTCCGCAC CACCGACGTC GGCCGCAAGC TGATCATCGA TCAGAACGTT 2161 TTTATCGAGG GTACGCTGCC GATGGGTGTC GTCCGCCCGC TGACTGAAGT CGAGATGGAC 2221 CATTACCGCG AGCCGTTCCT GAATCCTGTT GACCGCGAGC CACTGTGGCG CTTCCCAAAC 2281 GAGCTGCCAA TCGCCGGTGA GCCAGCGAAC ATCGTCGCGC TGGTCGAAGA ATACATGGAC 2341 TGGCTGCACC AGTCCCCTGT CCCGAAGCTG CTGTTCTGGG GCACCCCAGG CGTTCTGATC 2401 CCACCGGCCG AAGCCGCTCG CCTGGCCAAA AGCCTGCCTA ACTGCAAGGC TGTGGACATC 2461 GGCCCGGGTC TGAATCTGCT GCAAGAAGAC AACCCGGACC TGATCGGCAG CGAGATCGCG 2521 CGCTGGCTGT CGACGCTCGA GATTTCCGGC TCTAGAGAAG TGCACACAAA CCAGGACCCC 2581 CTGGACggat ccggatcagg atctggatca ggatcgggta gtgaacagaa gctgatcagc 2641 gaagaagacc tggagcaaaa gctcattagc gaggaggacc tcgagcagaa gctgattagc 2701 gaggaggacc tgtaaccacc accaccactg agaattctgt attacgccgt tttaagagct 2761 ccttcgacgc ctttttgtcc ttcagctgct ccacggtgac tcgcttctct ctctttccac 2821 agtgtctctt ttttcttttc actctctata caaatgtgag cgacctcctt ttctgtacaa 2881 cggccttccg gcgtgtgctt ttctcatcga cctctcctcg cttcttgggc actccttcat 2941 cgagcaaaca agacgaggga ggtgacgcga ttggtgagac cactacgagt atggacggag 3001 ctgtctgata ccccctgttt tttatcttgt accccgctct gcagtgacgg tagcccgctg 3061 ctgtgcttcg gtatcgccgc ttcatacgct tctctctttt ttcaacgtgc ggcgctgatt 3121 caaaggttac ctcaacgcga cccgccgctg catcctttgt tgctcctctt gtcgcaaaca 3181 aacaaaaaac gtgctgtgct ttttccttta tcgtgtcttt ttgcaaagtc tagagtttac 3241 cgacaagacc agaatagcgc ttatgtgtgt gtgtttgtgt gtgggtgtgt gtgcccatgt 3301 gtgacaaaga ggccagtgct ttctctaaag caagagcgcg aggttgctgg ctaaggactc 3361 taagtccctc acagtgatgg cgtgttgcat tttctaaacc gtcaatccaa cacgtcggta 3421 gcatatcggt tttgtaggct cttctggata ccttccccag taagaccgcc actgcttcgt 3481 gactgaagca ctgtaaatat cactgaaacc gtcgcgttgc tttgtctaat tatcaccgcc 3541 tttgccagct atcgcctcag gctgcgctct ttgactaatc cactcaccac ttctctgcct 3601 tcttcctccg gttgttgttg tgtcctgcgt gtacatggcg cgtgtccttt tcgagcaaac 3661 agctgtcttt tctttcacga taacacactc atattaaacg cgagtattac tcatcagtca 3721 acgtcacatt ccgctctgtc cacttcgacc ttacacctct acttgtccaa ctatcttcca 3781 cttgtcaagc ggctagcATG ACTGAATACA AGCCAACGGT TCGTCTTGCT ACGCGAGACG 3841 ATGTGCCGCG GGCCGTACGG ACGCTGGCTG CTGCGTTCGC TGATTATCCC GCCACCCGAC 3901 ATACGGTTGA CCCAGATCGA CACATTGAGC GAGTCACAGA ACTTCAAGAG TTATTCTTAA 3961 CACGGGTGGG CTTGGACATT GGTAAGGTTT GGGTTGCGGA CGATGGCGCG GCTGTTGCGG 4021 TGTGGACGAC GCCAGAGTCA GTCGAAGCGG GCGCAGTGTT TGCTGAGATC GGTCCCCGAA 4081 TGGCGGAGCT TAGCGGGTCA CGCCTCGCTG CACAACAGCA GATGGAAGGT CTGTTGGCAC 4141 CACATAGACC TAAGGAGCCG GCTTGGTTCT TAGCGACAGT TGGCGTCAGT CCGGACCACC 4201 AAGGTAAGGG GCTGGGTTCA GCGGTTGTTC TTCCAGGAGT AGAGGCTGCG GAAAGGGCAG 4261 GTGTGCCGGC GTTCCTTGAA ACGTCTGCAC CACGTAACCT TCCTTTTTAC GAGAGATTGG 4321 GGTTTACAGT AACAGCAGAT GTTGAAGTGC CTGAAGGGCC GAGGACATGG TGCATGACAC 4381 GTAAGCCGGG AGCCTAActc gagTGCCCAT TTAGTTGGCT TTTCCCTTGT CTCGTGTCTT 4441 TTCCGTGGAA AGGTTCCCGG AGTAATCTGA TGGCACAGCA GGGAGGTGCG CCTGCAGGTT 4501 GGTTAGGAAG GGGGGATGAT GTAAAAGAAG AAAATGGGGG GATTCGAGCC CCGCACAGGT 4561 CTCTCAAATT GGATGCATAG CTTGAGTATT CTATAGTGTC ACCTAAATAG CTTGGCGTAA 4621 TCATGGTCAT AGCTGTTTCC TGTGTGAAAT TGTTATCCGC TCACAATTCC ACACAACATA 4681 CGAGCCGGAA GCATAAAGTG TAAAGCCTGG GGTGCCTAAT GAGTGAGCTA ACTCACATTA 4741 ATTGCGTTGC GCTCACTGCC CGCTTTCCAG TCGGGAAACC TGTCGTGCCA GCTGCATTAA 4801 TGAATCGGCC AACGCGCGGG GAGAGGCGGT TTGCGTATTG GGCGCTCTTC CGCTTCCTCG 4861 CTCACTGACT CGCTGCGCTC GGTCGTTCGG CTGCGGCGAG CGGTATCAGC TCACTCAAAG 4921 GCGGTAATAC GGTTATCCAC AGAATCAGGG GATAACGCAG GAAAGAACAT GTGAGCAAAA 4981 GGCCAGCAAA AGGCCAGGAA CCGTAAAAAG GCCGCGTTGC TGGCGTTTTT CCATAGGCTC 5041 CGCCCCCCTG ACGAGCATCA CAAAAATCGA CGCTCAAGTC AGAGGTGGCG AAACCCGACA 5101 GGACTATAAA GATACCAGGC GTTTCCCCCT GGAAGCTCCC TCGTGCGCTC TCCTGTTCCG 5161 ACCCTGCCGC TTACCGGATA CCTGTCCGCC TTTCTCCCTT CGGGAAGCGT GGCGCTTTCT 5221 CATAGCTCAC GCTGTAGGTA TCTCAGTTCG GTGTAGGTCG TTCGCTCCAA GCTGGGCTGT 5281 GTGCACGAAC CCCCCGTTCA GCCCGACCGC TGCGCCTTAT CCGGTAACTA TCGTCTTGAG 5341 TCCAACCCGG TAAGACACGA CTTATCGCCA CTGGCAGCAG CCACTGGTAA CAGGATTAGC 5401 AGAGCGAGGT ATGTAGGCGG TGCTACAGAG TTCTTGAAGT GGTGGCCTAA CTACGGCTAC 5461 ACTAGAAGAA CAGTATTTGG TATCTGCGCT CTGCTGAAGC CAGTTACCTT CGGAAAAAGA 5521 GTTGGTAGCT CTTGATCCGG CAAACAAACC ACCGCTGGTA GCGGTGGTTT TTTTGTTTGC 5581 AAGCAGCAGA TTACGCGCAG AAAAAAAGGA TCTCAAGAAG ATCCTTTGAT CTTTTCTACG 5641 GGGTCTGACG CTCAGTGGAA CGAAAACTCA CGTTAAGGGA TTTTGGTCAT GAGATTATCA 5701 AAAAGGATCT TCACCTAGAT CCTTTTAAAT TAAAAATGAA GTTTTAAATC AATCTAAAGT 5761 ATATATGAGT AAACTTGGTC TGACAGTTAC CAATGCTTAA TCAGTGAGGC ACCTATCTCA 5821 GCGATCTGTC TATTTCGTTC ATCCATAGTT GCCTGACTCC CCGTCGTGTA GATAACTACG 5881 ATACGGGAGG GCTTACCATC TGGCCCCAGT GCTGCAATGA TACCGCGAGA CCCACGCTCA 5941 CCGGCTCCAG ATTTATCAGC AATAAACCAG CCAGCCGGAA GGGCCGAGCG CAGAAGTGGT 6001 CCTGCAACTT TATCCGCCTC CATCCAGTCT ATTAATTGTT GCCGGGAAGC TAGAGTAAGT 6061 AGTTCGCCAG TTAATAGTTT GCGCAACGTT GTTGCCATTG CTACAGGCAT CGTGGTGTCA 6121 CGCTCGTCGT TTGGTATGGC TTCATTCAGC TCCGGTTCCC AACGATCAAG GCGAGTTACA 6181 TGATCCCCCA TGTTGTGCAA AAAAGCGGTT AGCTCCTTCG GTCCTCCGAT CGTTGTCAGA 6241 AGTAAGTTGG CCGCAGTGTT ATCACTCATG GTTATGGCAG CACTGCATAA TTCTCTTACT 6301 GTCATGCCAT CCGTAAGATG CTTTTCTGTG ACTGGTGAGT ACTCAACCAA GTCATTCTGA 6361 GAATAGTGTA TGCGGCGACC GAGTTGCTCT TGCCCGGCGT CAATACGGGA TAATACCGCG 6421 CCACATAGCA GAACTTTAAA AGTGCTCATC ATTGGAAAAC GTTCTTCGGG GCGAAAACTC 6481 TCAAGGATCT TACCGCTGTT GAGATCCAGT TCGATGTAAC CCACTCGTGC ACCCAACTGA 6541 TCTTCAGCAT CTTTTACTTT CACCAGCGTT TCTGGGTGAG CAAAAACAGG AAGGCAAAAT 6601 GCCGCAAAAA AGGGAATAAG GGCGACACGG AAATGTTGAA TACTCATACT CTTCCTTTTT 6661 CAATATTATT GAAGCATTTA TCAGGGTTAT TGTCTCATGA GCGGATACAT ATTTGAATGT 6721 ATTTAGAAAA ATAAACAAAT AGGGGTTCCG CGCACATTTC CCCGAAAAGT GCCACCTGAT 6781 GCGGTGTGAA ATACCGCACA GATGCGTAAG GAGAAAATAC CGCATCAGGA AATTGTAAGC 6841 GTTAATATTT TGTTAAAATT CGCGTTAAAT TTTTGTTAAA TCAGCTCATT TTTTAACCAA 6901 TAGGCCGAAA TCGGCAAAAT CCCTTATAAA TCAAAAGAAT AGACCGAGAT AGGGTTGAGT 6961 GTTGTTCCAG TTTGGAACAA GAGTCCACTA TTAAAGAACG TGGACTCCAA CGTCAAAGGG 7021 CGAAAAACCG TCTATCAGGG CGATGGCCCA CTACGTGAAC CATCACCCTA ATCAAGTTTT 7081 TTGGGGTCGA GGTGCCGTAA AGCACTAAAT CGGAACCCTA AAGGGAGCCC CCGATTTAGA 7141 GCTTGACGGG GAAAGCCGGC GAACGTGGCG AGAAAGGAAG GGAAGAAAGC GAAAGGAGCG 7201 GGCGCTAGGG CGCTGGCAAG TGTAGCGGTC ACGCTGCGCG TAACCACCAC ACCCGCCGCG 7261 CTTAATGCGC CGCTACAGGG CGCGTCCATT CGCCATTCAG GCTGCGCAAC TGTTGGGAAG 7321 GGCGATCGGT GCGGGCCTCT TCGCTATTAC GCCAGCTGGC GAAAGGGGGA TGTGCTGCAA 7381 GGCGATTAAG TTGGGTAACG CCAGGGTTTT CCCAGTCACG ACGTTGTAAA ACGACGGCCA 7441 GTGAATTGTA ATACGACTCA CTATA // LOCUS pPLOT-mCH-Phleo-Phleo 6763 bp ds-DNA circular SYN 04-NOV-2016 DEFINITION pPLOT-mCH-Phleo-Phleo ACCESSION . VERSION . KEYWORDS Untitled SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6763) AUTHORS . TITLE Direct Submission JOURNAL Exported Friday, Nov 4, 2016 from SnapGene Viewer 3.2.1 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6763 /organism="synthetic DNA construct" /mol_type="other DNA" misc_feature 16..35 /note="#161" misc_feature 36..164 /note="Actin 5'" CDS 171..551 /codon_start=1 /note="BleoR" /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR EFALRDPAGNCVHFVAEEQDPW" misc_feature 558..706 /note="Ald3'UTR" misc_feature 713..1269 /note="PGKB 5'" CDS 1273..1362 /codon_start=1 /product="3 tandem Myc epitope tags" /note="3xMyc" /translation="EQKLISEEDLEQKLISEEDLEQKLISEEDL" misc_feature 1363..1392 /note="GS linker" misc_feature 1399..2103 /note="mCherry" misc_feature 2110..2139 /note="GS linker" CDS 2140..2229 /codon_start=1 /product="3 tandem Myc epitope tags" /note="3xMyc" /translation="EQKLISEEDLEQKLISEEDLEQKLISEEDL" misc_feature 2233..3308 /note="Cf PGKA/B" CDS 3315..3695 /codon_start=1 /note="BleoR" /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR EFALRDPAGNCVHFVAEEQDPW" misc_feature 3702..3850 /note="Ald3'UTR" promoter complement(3888..3906) /note="SP6 promoter" /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind complement(3924..3940) /note="M13 rev" /note="common sequencing primer, one of multiple similar variants" protein_bind 3948..3964 /bound_moiety="lac repressor encoded by lacI" /note="lac operator" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." promoter complement(3972..4002) /note="lac promoter" /note="promoter for the E. coli lac operon" protein_bind 4017..4038 /bound_moiety="E. coli catabolite activator protein" /note="CAP binding site" /note="CAP binding activates transcription in the presence of cAMP." rep_origin complement(4326..4914) /direction=LEFT /note="ori" /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(5085..5945) /codon_start=1 /gene="bla" /product="beta-lactamase" /note="AmpR" /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" promoter complement(5946..6050) /gene="bla" /note="AmpR promoter" rep_origin complement(6128..6583) /direction=LEFT /note="f1 ori" /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind 6724..6740 /note="M13 fwd" /note="common sequencing primer, one of multiple similar variants" promoter 6747..2 /note="T7 promoter" /note="promoter for bacteriophage T7 RNA polymerase" ORIGIN 1 gggcgaattg ggcccgtata atgcagacct gctgcaagct aggggcacag caaggtcttc 61 tgaaattcat gttttttttt tttttactct gcattgcagt ctccgctctt atttagtttt 121 gctttacgta aggtctcgtt gctgccataa aataagctct agtggaattc atggctaaac 181 tcacttctgc tgtaccagta ctcactgcta gagatgtagc tggagctgta gagttctgga 241 ctgatagact cggattctct agagatttcg tagaggatga tttcgctgga gtagtaagag 301 atgatgtaac tctcttcata tctgctgtac aagatcaagt agtaccagat aacactctcg 361 cttgggtatg ggtaagagga ctcgatgagc tctacgctga gtggtctgag gtagtatcta 421 ctaacttcag agatgcttct ggaccagcta tgactgagat aggagagcaa ccgtggggaa 481 gagagttcgc tctcagagat ccagctggaa actgcgtaca cttcgtagct gaggagcaag 541 atccatggta accatggtgc ccatttagtt ggcttttccc ttgtctcgtg tcttttccgt 601 ggaaaggttc ccggagtaat ctgatggcac agcagggagg tgcgcctgca ggttggttag 661 gaagggggga tgatgtaaaa gaagaaaatg gggggattcg agccccacgc gttcgagacc 721 gacaagacca gaatagcgct tatgtgtgtg tgtttgtgtg tgggtgtgtg tgcccatgtg 781 tgacaaagag gccagtgctt tctctaaagc aagagcgcga ggttgctggc taaggactct 841 aagtccctca cagtgatggc gtgttgcatt ttctaaaccg tcaatccaac acgtcggtag 901 catatcggtt ttgtaggctc ttctggatac cttccccagt aagaccgcca ctgcttcgtg 961 actgaagcac tgtaaatatc actgaaaccg tcgcgttgct ttgtctaatt atcaccgcct 1021 ttgccagcta tcgcctcagg ctgcgctctt tgactaatcc actcaccact tctctgcctt 1081 cttcctccgg ttgttgttgt gtcctgcgtg tacatggcgc gtgtcctttt cgagcaaaca 1141 gctgtctttt ctttcacgat aacacactca tattaaacgc gagtattact catcagtcaa 1201 cgtcacattc cgctctgtcc acttcgacct tacacctcta cttgtccaac tatcttccac 1261 ttgtcaagca tggaacagaa gctgatcagc gaagaagacc tggagcaaaa gctcattagc 1321 gaggaggacc tcgagcagaa gctgattagc gaggaggacc tgggttctgg tagtggttcc 1381 ggttccggtt ctaagcttgt tagtaaagga gaagaaaata acatggcaat cattaaggag 1441 ttcatgagat tcaaagttca catggaaggt tctgtaaatg gacatgaatt tgaaatagaa 1501 ggtgaaggag aaggaaggcc ttatgaagga acccaaaccg cgaagctaaa agttactaag 1561 ggtggcccat taccatttgc atgggatatc cttagccctc aattcatgta tgggtcaaag 1621 gcttatgtca agcaccccgc cgacattcca gactatctaa agttatcttt tcccgaaggg 1681 tttaagtggg agcgtgtgat gaacttcgaa gacggtggcg tggtaacagt gactcaggat 1741 tcgtccctgc aagatggtga atttatctac aaagtcaaat taagaggaac taactttcca 1801 tctgacggcc cggttatgca aaaaaagaca atgggctggg aggcctcctc agaacgaatg 1861 taccctgaag atggtgcctt gaagggtgag attaaacaaa gattgaaatt gaaagatggt 1921 ggacattatg acgctgaggt taaaacgaca tacaaagcta agaaacctgt ccagctccca 1981 ggtgcttaca atgtaaatat aaaacttgat attacatcac ataatgaaga ttatacgata 2041 gttgaacaat acgaaagggc tgagggagac atagtactgg tggcatggat gaactataca 2101 aagggatccg gatcaggatc tggatcagga tcgggtagtg aacagaagct gatcagcgaa 2161 gaagacctgg agcaaaagct cattagcgag gaggacctcg agcagaagct gattagcgag 2221 gaggacctgt aaccaccacc accactgaga attctgtatt acgccgtttt aagagctcct 2281 tcgacgcctt tttgtccttc agctgctcca cggtgactcg cttctctctc tttccacagt 2341 gtctcttttt tcttttcact ctctatacaa atgtgagcga cctccttttc tgtacaacgg 2401 ccttccggcg tgtgcttttc tcatcgacct ctcctcgctt cttgggcact ccttcatcga 2461 gcaaacaaga cgagggaggt gacgcgattg gtgagaccac tacgagtatg gacggagctg 2521 tctgataccc cctgtttttt atcttgtacc ccgctctgca gtgacggtag cccgctgctg 2581 tgcttcggta tcgccgcttc atacgcttct ctcttttttc aacgtgcggc gctgattcaa 2641 aggttacctc aacgcgaccc gccgctgcat cctttgttgc tcctcttgtc gcaaacaaac 2701 aaaaaacgtg ctgtgctttt tcctttatcg tgtctttttg caaagtctag agtttaccga 2761 caagaccaga atagcgctta tgtgtgtgtg tttgtgtgtg ggtgtgtgtg cccatgtgtg 2821 acaaagaggc cagtgctttc tctaaagcaa gagcgcgagg ttgctggcta aggactctaa 2881 gtccctcaca gtgatggcgt gttgcatttt ctaaaccgtc aatccaacac gtcggtagca 2941 tatcggtttt gtaggctctt ctggatacct tccccagtaa gaccgccact gcttcgtgac 3001 tgaagcactg taaatatcac tgaaaccgtc gcgttgcttt gtctaattat caccgccttt 3061 gccagctatc gcctcaggct gcgctctttg actaatccac tcaccacttc tctgccttct 3121 tcctccggtt gttgttgtgt cctgcgtgta catggcgcgt gtccttttcg agcaaacagc 3181 tgtcttttct ttcacgataa cacactcata ttaaacgcga gtattactca tcagtcaacg 3241 tcacattccg ctctgtccac ttcgacctta cacctctact tgtccaacta tcttccactt 3301 gtcaagcggc tagcatggct aaactcactt ctgctgtacc agtactcact gctagagatg 3361 tagctggagc tgtagagttc tggactgata gactcggatt ctctagagat ttcgtagagg 3421 atgatttcgc tggagtagta agagatgatg taactctctt catatctgct gtacaagatc 3481 aagtagtacc agataacact ctcgcttggg tatgggtaag aggactcgat gagctctacg 3541 ctgagtggtc tgaggtagta tctactaact tcagagatgc ttctggacca gctatgactg 3601 agataggaga gcaaccgtgg ggaagagagt tcgctctcag agatccagct ggaaactgcg 3661 tacacttcgt agctgaggag caagatccat ggtaactcga gtgcccattt agttggcttt 3721 tcccttgtct cgtgtctttt ccgtggaaag gttcccggag taatctgatg gcacagcagg 3781 gaggtgcgcc tgcaggttgg ttaggaaggg gggatgatgt aaaagaagaa aatgggggga 3841 ttcgagcccc gcacaggtct ctcaaattgg atgcatagct tgagtattct atagtgtcac 3901 ctaaatagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc 3961 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga 4021 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg 4081 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg 4141 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg 4201 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga 4261 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg 4321 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag 4381 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc 4441 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg 4501 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt 4561 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc 4621 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc 4681 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg 4741 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca 4801 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc 4861 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat 4921 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt 4981 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt 5041 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc 5101 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc 5161 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata 5221 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg 5281 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc 5341 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct 5401 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa 5461 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt 5521 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca 5581 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac 5641 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca 5701 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt 5761 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc 5821 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca 5881 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata 5941 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc 6001 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc 6061 cgaaaagtgc cacctgatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg 6121 catcaggaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc 6181 agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag 6241 accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg 6301 gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca 6361 tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa 6421 gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg 6481 aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta 6541 accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtccattcg ccattcaggc 6601 tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga 6661 aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac 6721 gttgtaaaac gacggccagt gaattgtaat acgactcact ata // LOCUS pPLOT-mCH-Puro-Puro 7201 bp ds-DNA circular 24-AUG-2016 DEFINITION pPLOT-mCH-Puro-Puro FEATURES Location/Qualifiers rep_origin complement(4746..5428) /label="ColE1 origin" /ApEinfo_revcolor=#ff9ccd /ApEinfo_fwdcolor=#ff9ccd misc_feature 932..1488 /label="PGKB 5'" /ApEinfo_revcolor=#faac61 /ApEinfo_fwdcolor=#faac61 CDS 1492..1581 /label="3xMyc" /ApEinfo_revcolor=#ffef86 /ApEinfo_fwdcolor=#ffef86 primer_bind 1..3 /label="T7" /ApEinfo_revcolor=#d59687 /ApEinfo_fwdcolor=#d59687 CDS complement(5526..6185) /label="AmpR" /ApEinfo_revcolor=#d6b295 /ApEinfo_fwdcolor=#d6b295 primer_bind 7161..7178 /label="M13-fwd" /ApEinfo_revcolor=#ffef86 /ApEinfo_fwdcolor=#ffef86 rep_origin complement(6698..7004) /label="F1 ori" /ApEinfo_revcolor=#75c6a9 /ApEinfo_fwdcolor=#75c6a9 primer_bind complement(4327..4352) /label="SP6" /ApEinfo_revcolor=#84b0dc /ApEinfo_fwdcolor=#84b0dc primer_bind 7177..3 /label="T7" /ApEinfo_revcolor=#85dae9 /ApEinfo_fwdcolor=#85dae9 misc_feature 3534..4133 /label="PuroR" /ApEinfo_revcolor=#d59687 /ApEinfo_fwdcolor=#d59687 primer_bind 16..35 /label="#161" /ApEinfo_revcolor=#f58a5e /ApEinfo_fwdcolor=#f58a5e misc_binding complement(4384..4406) /label="LacO" /ApEinfo_revcolor=#f8d3a9 /ApEinfo_fwdcolor=#f8d3a9 5'UTR 36..164 /label="Actin 5'" /ApEinfo_revcolor=#b7e6d7 /ApEinfo_fwdcolor=#b7e6d7 CDS 2359..2448 /label="3xMyc" /ApEinfo_revcolor=#ffef86 /ApEinfo_fwdcolor=#ffef86 misc_feature 1582..1601 /label="Primer C-terminal For" /ApEinfo_revcolor=#ff9ccd /ApEinfo_fwdcolor=#ff9ccd misc_feature complement(2339..2358) /label="Primer N-terminal Rev" /ApEinfo_revcolor=#9eafd2 /ApEinfo_fwdcolor=#9eafd2 primer_bind 7177..7201 /label="T7" /ApEinfo_revcolor=#b7e6d7 /ApEinfo_fwdcolor=#b7e6d7 misc_feature 2329..2358 /label="GS linker" /ApEinfo_revcolor=#d59687 /ApEinfo_fwdcolor=#d59687 CDS complement(7022..7090) /label="LacZ alpha" /ApEinfo_revcolor=#b4abac /ApEinfo_fwdcolor=#b4abac misc_feature 1582..1611 /label="GS linker" /ApEinfo_revcolor=#84b0dc /ApEinfo_fwdcolor=#84b0dc primer_bind complement(4358..4378) /label="M13-rev" /ApEinfo_revcolor=#b7e6d7 /ApEinfo_fwdcolor=#b7e6d7 misc_feature 777..925 /label="Ald3'UTR" /ApEinfo_revcolor=#b1ff67 /ApEinfo_fwdcolor=#b1ff67 3'UTR 4140..4288 /label="Ald3'UTR (short)(1)" /ApEinfo_revcolor=#c7b0e3 /ApEinfo_fwdcolor=#c7b0e3 misc_feature 171..770 /label="PuroR" /ApEinfo_revcolor=#d59687 /ApEinfo_fwdcolor=#d59687 CDS 1618..2322 /label="mCherry - codon optimised" /ApEinfo_revcolor=#9eafd2 /ApEinfo_fwdcolor=#9eafd2 misc_feature 2452..3527 /label="Cf PGKA/B" /ApEinfo_revcolor=#c7b0e3 /ApEinfo_fwdcolor=#c7b0e3 primer_bind complement(4289..4308) /label="#162" /ApEinfo_revcolor=#f58a5e /ApEinfo_fwdcolor=#f58a5e ORIGIN 1 GGGCGAATTG GGCCCgtata atgcagacct gctgcAAGCT AGGGGCACAG CAAGGTCTTC 61 TGAAATTCAT GTTTTTTTTT TTTTTACTCT GCATTGCAGT CTCCGCTCTT ATTTAGTTTT 121 GCTTTACGTA AGGTCTCGTT GCTGCCATAA AATAAGCTCT AGTGgaattc ATGACTGAAT 181 ACAAGCCAAC GGTTCGTCTT GCTACGCGAG ACGATGTGCC GCGGGCCGTA CGGACGCTGG 241 CTGCTGCGTT CGCTGATTAT CCCGCCACCC GACATACGGT TGACCCAGAT CGACACATTG 301 AGCGAGTCAC AGAACTTCAA GAGTTATTCT TAACACGGGT GGGCTTGGAC ATTGGTAAGG 361 TTTGGGTTGC GGACGATGGC GCGGCTGTTG CGGTGTGGAC GACGCCAGAG TCAGTCGAAG 421 CGGGCGCAGT GTTTGCTGAG ATCGGTCCCC GAATGGCGGA GCTTAGCGGG TCACGCCTCG 481 CTGCACAACA GCAGATGGAA GGTCTGTTGG CACCACATAG ACCTAAGGAG CCGGCTTGGT 541 TCTTAGCGAC AGTTGGCGTC AGTCCGGACC ACCAAGGTAA GGGGCTGGGT TCAGCGGTTG 601 TTCTTCCAGG AGTAGAGGCT GCGGAAAGGG CAGGTGTGCC GGCGTTCCTT GAAACGTCTG 661 CACCACGTAA CCTTCCTTTT TACGAGAGAT TGGGGTTTAC AGTAACAGCA GATGTTGAAG 721 TGCCTGAAGG GCCGAGGACA TGGTGCATGA CACGTAAGCC GGGAGCCTAA ccatggtgcc 781 catttagttg gcttttccct tgtctcgtgt cttttccgtg gaaaggttcc cggagtaatc 841 tgatggcaca gcagggaggt gcgcctgcag gttggttagg aaggggggat gatgtaaaag 901 aagaaaatgg ggggattcga gccccacgcg ttcgagaccg acaagaccag aatagcgctt 961 atgtgtgtgt gtttgtgtgt gggtgtgtgt gcccatgtgt gacaaagagg ccagtgcttt 1021 ctctaaagca agagcgcgag gttgctggct aaggactcta agtccctcac agtgatggcg 1081 tgttgcattt tctaaaccgt caatccaaca cgtcggtagc atatcggttt tgtaggctct 1141 tctggatacc ttccccagta agaccgccac tgcttcgtga ctgaagcact gtaaatatca 1201 ctgaaaccgt cgcgttgctt tgtctaatta tcaccgcctt tgccagctat cgcctcaggc 1261 tgcgctcttt gactaatcca ctcaccactt ctctgccttc ttcctccggt tgttgttgtg 1321 tcctgcgtgt acatggcgcg tgtccttttc gagcaaacag ctgtcttttc tttcacgata 1381 acacactcat attaaacgcg agtattactc atcagtcaac gtcacattcc gctctgtcca 1441 cttcgacctt acacctctac ttgtccaact atcttccact tgtcaagcat ggaacagaag 1501 ctgatcagcg aagaagacct ggagcaaaag ctcattagcg aggaggacct cgagcagaag 1561 ctgattagcg aggaggacct gggttctggt agtggttccg gttccggttc taagcttGTT 1621 AGTAAAGGAG AAGAAAATAA CATGGCAATC ATTAAGGAGT TCATGAGATT CAAAGTTCAC 1681 ATGGAAGGTT CTGTAAATGG ACATGAATTT GAAATAGAAG GTGAAGGAGA AGGAAGGCCT 1741 TATGAAGGAA CCCAAACCGC GAAGCTAAAA GTTACTAAGG GTGGCCCATT ACCATTTGCA 1801 TGGGATATCC TTAGCCCTCA ATTCATGTAT GGGTCAAAGG CTTATGTCAA GCACCCCGCC 1861 GACATTCCAG ACTATCTAAA GTTATCTTTT CCCGAAGGGT TTAAGTGGGA GCGTGTGATG 1921 AACTTCGAAG ACGGTGGCGT GGTAACAGTG ACTCAGGATT CGTCCCTGCA AGATGGTGAA 1981 TTTATCTACA AAGTCAAATT AAGAGGAACT AACTTTCCAT CTGACGGCCC GGTTATGCAA 2041 AAAAAGACAA TGGGCTGGGA GGCCTCCTCA GAACGAATGT ACCCTGAAGA TGGTGCCTTG 2101 AAGGGTGAGA TTAAACAAAG ATTGAAATTG AAAGATGGTG GACATTATGA CGCTGAgGTT 2161 AAAACGACAT ACAAAGCTAA GAAACCTGTC CAGCTCCCAG GTGCTTACAA TGTAAATATA 2221 AAACTTGATA TTACATCACA TAATGAAGAT TATACGATAG TTGAACAATA CGAAAGGGcT 2281 GAGGGAGACA TAGTACTGGT GGCATGGaTG AACTATACAA AGggatccgg atcaggatct 2341 ggatcaggat cgggtagtga acagaagctg atcagcgaag aagacctgga gcaaaagctc 2401 attagcgagg aggacctcga gcagaagctg attagcgagg aggacctgta accaccacca 2461 ccactgagaa ttctgtatta cgccgtttta agagctcctt cgacgccttt ttgtccttca 2521 gctgctccac ggtgactcgc ttctctctct ttccacagtg tctctttttt cttttcactc 2581 tctatacaaa tgtgagcgac ctccttttct gtacaacggc cttccggcgt gtgcttttct 2641 catcgacctc tcctcgcttc ttgggcactc cttcatcgag caaacaagac gagggaggtg 2701 acgcgattgg tgagaccact acgagtatgg acggagctgt ctgatacccc ctgtttttta 2761 tcttgtaccc cgctctgcag tgacggtagc ccgctgctgt gcttcggtat cgccgcttca 2821 tacgcttctc tcttttttca acgtgcggcg ctgattcaaa ggttacctca acgcgacccg 2881 ccgctgcatc ctttgttgct cctcttgtcg caaacaaaca aaaaacgtgc tgtgcttttt 2941 cctttatcgt gtctttttgc aaagtctaga gtttaccgac aagaccagaa tagcgcttat 3001 gtgtgtgtgt ttgtgtgtgg gtgtgtgtgc ccatgtgtga caaagaggcc agtgctttct 3061 ctaaagcaag agcgcgaggt tgctggctaa ggactctaag tccctcacag tgatggcgtg 3121 ttgcattttc taaaccgtca atccaacacg tcggtagcat atcggttttg taggctcttc 3181 tggatacctt ccccagtaag accgccactg cttcgtgact gaagcactgt aaatatcact 3241 gaaaccgtcg cgttgctttg tctaattatc accgcctttg ccagctatcg cctcaggctg 3301 cgctctttga ctaatccact caccacttct ctgccttctt cctccggttg ttgttgtgtc 3361 ctgcgtgtac atggcgcgtg tccttttcga gcaaacagct gtcttttctt tcacgataac 3421 acactcatat taaacgcgag tattactcat cagtcaacgt cacattccgc tctgtccact 3481 tcgaccttac acctctactt gtccaactat cttccacttg tcaagcggct agcATGACTG 3541 AATACAAGCC AACGGTTCGT CTTGCTACGC GAGACGATGT GCCGCGGGCC GTACGGACGC 3601 TGGCTGCTGC GTTCGCTGAT TATCCCGCCA CCCGACATAC GGTTGACCCA GATCGACACA 3661 TTGAGCGAGT CACAGAACTT CAAGAGTTAT TCTTAACACG GGTGGGCTTG GACATTGGTA 3721 AGGTTTGGGT TGCGGACGAT GGCGCGGCTG TTGCGGTGTG GACGACGCCA GAGTCAGTCG 3781 AAGCGGGCGC AGTGTTTGCT GAGATCGGTC CCCGAATGGC GGAGCTTAGC GGGTCACGCC 3841 TCGCTGCACA ACAGCAGATG GAAGGTCTGT TGGCACCACA TAGACCTAAG GAGCCGGCTT 3901 GGTTCTTAGC GACAGTTGGC GTCAGTCCGG ACCACCAAGG TAAGGGGCTG GGTTCAGCGG 3961 TTGTTCTTCC AGGAGTAGAG GCTGCGGAAA GGGCAGGTGT GCCGGCGTTC CTTGAAACGT 4021 CTGCACCACG TAACCTTCCT TTTTACGAGA GATTGGGGTT TACAGTAACA GCAGATGTTG 4081 AAGTGCCTGA AGGGCCGAGG ACATGGTGCA TGACACGTAA GCCGGGAGCC TAActcgagT 4141 GCCCATTTAG TTGGCTTTTC CCTTGTCTCG TGTCTTTTCC GTGGAAAGGT TCCCGGAGTA 4201 ATCTGATGGC ACAGCAGGGA GGTGCGCCTG CAGGTTGGTT AGGAAGGGGG GATGATGTAA 4261 AAGAAGAAAA TGGGGGGATT CGAGCCCCGC ACAGGTCTCT CAAATTGGAT GCATAGCTTG 4321 AGTATTCTAT AGTGTCACCT AAATAGCTTG GCGTAATCAT GGTCATAGCT GTTTCCTGTG 4381 TGAAATTGTT ATCCGCTCAC AATTCCACAC AACATACGAG CCGGAAGCAT AAAGTGTAAA 4441 GCCTGGGGTG CCTAATGAGT GAGCTAACTC ACATTAATTG CGTTGCGCTC ACTGCCCGCT 4501 TTCCAGTCGG GAAACCTGTC GTGCCAGCTG CATTAATGAA TCGGCCAACG CGCGGGGAGA 4561 GGCGGTTTGC GTATTGGGCG CTCTTCCGCT TCCTCGCTCA CTGACTCGCT GCGCTCGGTC 4621 GTTCGGCTGC GGCGAGCGGT ATCAGCTCAC TCAAAGGCGG TAATACGGTT ATCCACAGAA 4681 TCAGGGGATA ACGCAGGAAA GAACATGTGA GCAAAAGGCC AGCAAAAGGC CAGGAACCGT 4741 AAAAAGGCCG CGTTGCTGGC GTTTTTCCAT AGGCTCCGCC CCCCTGACGA GCATCACAAA 4801 AATCGACGCT CAAGTCAGAG GTGGCGAAAC CCGACAGGAC TATAAAGATA CCAGGCGTTT 4861 CCCCCTGGAA GCTCCCTCGT GCGCTCTCCT GTTCCGACCC TGCCGCTTAC CGGATACCTG 4921 TCCGCCTTTC TCCCTTCGGG AAGCGTGGCG CTTTCTCATA GCTCACGCTG TAGGTATCTC 4981 AGTTCGGTGT AGGTCGTTCG CTCCAAGCTG GGCTGTGTGC ACGAACCCCC CGTTCAGCCC 5041 GACCGCTGCG CCTTATCCGG TAACTATCGT CTTGAGTCCA ACCCGGTAAG ACACGACTTA 5101 TCGCCACTGG CAGCAGCCAC TGGTAACAGG ATTAGCAGAG CGAGGTATGT AGGCGGTGCT 5161 ACAGAGTTCT TGAAGTGGTG GCCTAACTAC GGCTACACTA GAAGAACAGT ATTTGGTATC 5221 TGCGCTCTGC TGAAGCCAGT TACCTTCGGA AAAAGAGTTG GTAGCTCTTG ATCCGGCAAA 5281 CAAACCACCG CTGGTAGCGG TGGTTTTTTT GTTTGCAAGC AGCAGATTAC GCGCAGAAAA 5341 AAAGGATCTC AAGAAGATCC TTTGATCTTT TCTACGGGGT CTGACGCTCA GTGGAACGAA 5401 AACTCACGTT AAGGGATTTT GGTCATGAGA TTATCAAAAA GGATCTTCAC CTAGATCCTT 5461 TTAAATTAAA AATGAAGTTT TAAATCAATC TAAAGTATAT ATGAGTAAAC TTGGTCTGAC 5521 AGTTACCAAT GCTTAATCAG TGAGGCACCT ATCTCAGCGA TCTGTCTATT TCGTTCATCC 5581 ATAGTTGCCT GACTCCCCGT CGTGTAGATA ACTACGATAC GGGAGGGCTT ACCATCTGGC 5641 CCCAGTGCTG CAATGATACC GCGAGACCCA CGCTCACCGG CTCCAGATTT ATCAGCAATA 5701 AACCAGCCAG CCGGAAGGGC CGAGCGCAGA AGTGGTCCTG CAACTTTATC CGCCTCCATC 5761 CAGTCTATTA ATTGTTGCCG GGAAGCTAGA GTAAGTAGTT CGCCAGTTAA TAGTTTGCGC 5821 AACGTTGTTG CCATTGCTAC AGGCATCGTG GTGTCACGCT CGTCGTTTGG TATGGCTTCA 5881 TTCAGCTCCG GTTCCCAACG ATCAAGGCGA GTTACATGAT CCCCCATGTT GTGCAAAAAA 5941 GCGGTTAGCT CCTTCGGTCC TCCGATCGTT GTCAGAAGTA AGTTGGCCGC AGTGTTATCA 6001 CTCATGGTTA TGGCAGCACT GCATAATTCT CTTACTGTCA TGCCATCCGT AAGATGCTTT 6061 TCTGTGACTG GTGAGTACTC AACCAAGTCA TTCTGAGAAT AGTGTATGCG GCGACCGAGT 6121 TGCTCTTGCC CGGCGTCAAT ACGGGATAAT ACCGCGCCAC ATAGCAGAAC TTTAAAAGTG 6181 CTCATCATTG GAAAACGTTC TTCGGGGCGA AAACTCTCAA GGATCTTACC GCTGTTGAGA 6241 TCCAGTTCGA TGTAACCCAC TCGTGCACCC AACTGATCTT CAGCATCTTT TACTTTCACC 6301 AGCGTTTCTG GGTGAGCAAA AACAGGAAGG CAAAATGCCG CAAAAAAGGG AATAAGGGCG 6361 ACACGGAAAT GTTGAATACT CATACTCTTC CTTTTTCAAT ATTATTGAAG CATTTATCAG 6421 GGTTATTGTC TCATGAGCGG ATACATATTT GAATGTATTT AGAAAAATAA ACAAATAGGG 6481 GTTCCGCGCA CATTTCCCCG AAAAGTGCCA CCTGATGCGG TGTGAAATAC CGCACAGATG 6541 CGTAAGGAGA AAATACCGCA TCAGGAAATT GTAAGCGTTA ATATTTTGTT AAAATTCGCG 6601 TTAAATTTTT GTTAAATCAG CTCATTTTTT AACCAATAGG CCGAAATCGG CAAAATCCCT 6661 TATAAATCAA AAGAATAGAC CGAGATAGGG TTGAGTGTTG TTCCAGTTTG GAACAAGAGT 6721 CCACTATTAA AGAACGTGGA CTCCAACGTC AAAGGGCGAA AAACCGTCTA TCAGGGCGAT 6781 GGCCCACTAC GTGAACCATC ACCCTAATCA AGTTTTTTGG GGTCGAGGTG CCGTAAAGCA 6841 CTAAATCGGA ACCCTAAAGG GAGCCCCCGA TTTAGAGCTT GACGGGGAAA GCCGGCGAAC 6901 GTGGCGAGAA AGGAAGGGAA GAAAGCGAAA GGAGCGGGCG CTAGGGCGCT GGCAAGTGTA 6961 GCGGTCACGC TGCGCGTAAC CACCACACCC GCCGCGCTTA ATGCGCCGCT ACAGGGCGCG 7021 TCCATTCGCC ATTCAGGCTG CGCAACTGTT GGGAAGGGCG ATCGGTGCGG GCCTCTTCGC 7081 TATTACGCCA GCTGGCGAAA GGGGGATGTG CTGCAAGGCG ATTAAGTTGG GTAACGCCAG 7141 GGTTTTCCCA GTCACGACGT TGTAAAACGA CGGCCAGTGA ATTGTAATAC GACTCACTAT 7201 A // LOCUS pPLOT-mNG-blast-blast 6787 bp ds-DNA circular 03-JUN-2016 DEFINITION pPLOT-mNG-blast-blast SOURCE synthetic DNA construct ORGANISM synthetic DNA construct FEATURES Location/Qualifiers protein_bind 4041..4062 /label="CAP binding activates transcription in the presence of cAMP." /ApEinfo_revcolor=#c7b0e3 /ApEinfo_fwdcolor=#c7b0e3 promoter 6771..2 /label="promoter for bacteriophage T7 RNA polymerase" /ApEinfo_revcolor=#faac61 /ApEinfo_fwdcolor=#faac61 CDS 1285..1374 /label="3xMyc" /ApEinfo_revcolor=#ffef86 /ApEinfo_fwdcolor=#ffef86 misc_feature 1375..1394 /label="Primer C-terminal For" /ApEinfo_revcolor=#ff9ccd /ApEinfo_fwdcolor=#ff9ccd primer_bind 6748..6764 /label="common sequencing primer, one of multiple similar variants" /ApEinfo_revcolor=#f8d3a9 /ApEinfo_fwdcolor=#f8d3a9 promoter complement(5970..6074) /label="bla promoter" /ApEinfo_revcolor=#b4abac /ApEinfo_fwdcolor=#b4abac misc_feature 171..560 /label="BlastR" /ApEinfo_revcolor=#d59687 /ApEinfo_fwdcolor=#d59687 misc_feature complement(2132..2151) /label="Primer N-terminal Rev" /ApEinfo_revcolor=#9eafd2 /ApEinfo_fwdcolor=#9eafd2 CDS complement(5112..5771) /label="AmpR" /ApEinfo_revcolor=#ffef86 /ApEinfo_fwdcolor=#ffef86 misc_feature 3726..3874 /label="Ald3'UTR" /ApEinfo_revcolor=#ff9ccd /ApEinfo_fwdcolor=#ff9ccd misc_feature 1375..1404 /label="GS linker" /ApEinfo_revcolor=#84b0dc /ApEinfo_fwdcolor=#84b0dc misc_feature 36..164 /label="Actin 5'" /ApEinfo_revcolor=#b4abac /ApEinfo_fwdcolor=#b4abac misc_feature 725..1281 /label="PGKB 5'" /ApEinfo_revcolor=#faac61 /ApEinfo_fwdcolor=#faac61 misc_feature 570..718 /label="Ald3'UTR" /ApEinfo_revcolor=#b1ff67 /ApEinfo_fwdcolor=#b1ff67 misc_feature complement(3875..3894) /label="#162" /ApEinfo_revcolor=#ff9ccd /ApEinfo_fwdcolor=#ff9ccd misc_feature 3327..3716 /label="BlastR" /ApEinfo_revcolor=#d59687 /ApEinfo_fwdcolor=#d59687 CDS 2152..2241 /label="3xMyc" /ApEinfo_revcolor=#ffef86 /ApEinfo_fwdcolor=#ffef86 misc_feature 2122..2151 /label="GS linker" /ApEinfo_revcolor=#d59687 /ApEinfo_fwdcolor=#d59687 misc_feature complement(3875..3894) /label="Primer C-terminal Rev" /ApEinfo_revcolor=#faac61 /ApEinfo_fwdcolor=#faac61 misc_feature 16..35 /label="Primer N-terminal For" /ApEinfo_revcolor=#9eafd2 /ApEinfo_fwdcolor=#9eafd2 promoter complement(3996..4026) /label="promoter for the E. coli lac operon" /ApEinfo_revcolor=#c7b0e3 /ApEinfo_fwdcolor=#c7b0e3 promoter complement(3912..3930) /label="promoter for bacteriophage SP6 RNA polymerase" /ApEinfo_revcolor=#ffef86 /ApEinfo_fwdcolor=#ffef86 primer_bind complement(3913..3938) /label="SP6" /ApEinfo_revcolor=#85dae9 /ApEinfo_fwdcolor=#85dae9 misc_feature 1411..2115 /label="mNeonGreen" /ApEinfo_revcolor=#b1ff67 /ApEinfo_fwdcolor=#b1ff67 rep_origin complement(6152..6607) /label="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" /ApEinfo_revcolor=#9eafd2 /ApEinfo_fwdcolor=#9eafd2 protein_bind 3972..3988 /label="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." /ApEinfo_revcolor=#ff9ccd /ApEinfo_fwdcolor=#ff9ccd primer_bind complement(3948..3964) /label="common sequencing primer, one of multiple similar variants" /ApEinfo_revcolor=#f8d3a9 /ApEinfo_fwdcolor=#f8d3a9 misc_feature 2245..3320 /label="Cf PGKA/B" /ApEinfo_revcolor=#c7b0e3 /ApEinfo_fwdcolor=#c7b0e3 misc_feature 16..35 /label="#161" /ApEinfo_revcolor=#d59687 /ApEinfo_fwdcolor=#d59687 primer_bind 6763..3 /label="T7" /ApEinfo_revcolor=#85dae9 /ApEinfo_fwdcolor=#85dae9 rep_origin complement(4350..4938) /label="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" /ApEinfo_revcolor=#c7b0e3 /ApEinfo_fwdcolor=#c7b0e3 ORIGIN 1 gggcgaattg ggcccgtata atgcagacct gctgcaagct aggggcacag caaggtcttc 61 tgaaattcat gttttttttt tttttactct gcattgcagt ctccgctctt atttagtttt 121 gctttacgta aggtctcgtt gctgccataa aataagctct agtggaattc atgcctttgt 181 ctcaagaaga atccaccctc attgaaagag caacggctac aatcaacagc atccccatct 241 ctgaagacta cagcgtcgcc agcgcagctc tctctagcga cggccgcatc ttcactggtg 301 tcaatgtata tcattttact gggggacctt gtgcagaact cgtggtgctg ggcactgctg 361 ctgctgcggc agctggcaac ctgacttgta tcgtcgcgat cggaaatgag aacaggggca 421 tcttgagccc ctgcggacgg tgccgacagg tgcttctcga tctgcatcct gggatcaaag 481 ccatagtgaa ggacagtgat ggacagccga cggcagttgg gattcgtgaa ttgctgccct 541 ctggttatgt gtgggagggc taaccatggt gcccatttag ttggcttttc ccttgtctcg 601 tgtcttttcc gtggaaaggt tcccggagta atctgatggc acagcaggga ggtgcgcctg 661 caggttggtt aggaaggggg gatgatgtaa aagaagaaaa tggggggatt cgagccccac 721 gcgttcgaga ccgacaagac cagaatagcg cttatgtgtg tgtgtttgtg tgtgggtgtg 781 tgtgcccatg tgtgacaaag aggccagtgc tttctctaaa gcaagagcgc gaggttgctg 841 gctaaggact ctaagtccct cacagtgatg gcgtgttgca ttttctaaac cgtcaatcca 901 acacgtcggt agcatatcgg ttttgtaggc tcttctggat accttcccca gtaagaccgc 961 cactgcttcg tgactgaagc actgtaaata tcactgaaac cgtcgcgttg ctttgtctaa 1021 ttatcaccgc ctttgccagc tatcgcctca ggctgcgctc tttgactaat ccactcacca 1081 cttctctgcc ttcttcctcc ggttgttgtt gtgtcctgcg tgtacatggc gcgtgtcctt 1141 ttcgagcaaa cagctgtctt ttctttcacg ataacacact catattaaac gcgagtatta 1201 ctcatcagtc aacgtcacat tccgctctgt ccacttcgac cttacacctc tacttgtcca 1261 actatcttcc acttgtcaag catggaacag aagctgatca gcgaagaaga cctggagcaa 1321 aagctcatta gcgaggagga cctcgagcag aagctgatta gcgaggagga cctgggttct 1381 ggtagtggtt ccggttccgg ttctaagctt gtctcgaaag gtgaggaaga caatatggcc 1441 agcttgccag caacgcacga gcttcatata tttgggtcga tcaacggtgt ggatttcgac 1501 atggtgggtc aaggcacagg taatcctaat gacgggtatg aagaactcaa tttgaagagc 1561 actaagggag atctccagtt ttccccgtgg attcttgtgc cgcatattgg ttatggtttt 1621 catcagtact taccataccc cgatggaatg tcgcccttcc aagcagctat ggtggacggc 1681 tcggggtatc aagtacatcg caccatgcaa tttgaggacg gagcatccct caccgtaaac 1741 taccgctata cgtatgaggg gagccacata aaaggtgagg ctcaagttaa aggcacaggc 1801 ttcccagctg acggcccggt catgaccaat tcattaaccg ctgctgattg gtgccgttct 1861 aagaagacct accctaacga caagacgatc attagtactt ttaagtggag ttataccaca 1921 ggaaacggaa agcggtatcg aagcactgct cgaacaacct acacatttgc gaagccaatg 1981 gccgcgaact acttgaagaa ccagccaatg tatgtgtttc gaaaaacgga actgaagcat 2041 tcgaaaaccg agcttaattt taaagagtgg cagaaggcgt ttacagacgt gatggggatg 2101 gacgaattgt ataagggatc cggatcagga tctggatcag gatcgggtag tgaacagaag 2161 ctgatcagcg aagaagacct ggagcaaaag ctcattagcg aggaggacct cgagcagaag 2221 ctgattagcg aggaggacct gtaaccacca ccaccactga gaattctgta ttacgccgtt 2281 ttaagagctc cttcgacgcc tttttgtcct tcagctgctc cacggtgact cgcttctctc 2341 tctttccaca gtgtctcttt tttcttttca ctctctatac aaatgtgagc gacctccttt 2401 tctgtacaac ggccttccgg cgtgtgcttt tctcatcgac ctctcctcgc ttcttgggca 2461 ctccttcatc gagcaaacaa gacgagggag gtgacgcgat tggtgagacc actacgagta 2521 tggacggagc tgtctgatac cccctgtttt ttatcttgta ccccgctctg cagtgacggt 2581 agcccgctgc tgtgcttcgg tatcgccgct tcatacgctt ctctcttttt tcaacgtgcg 2641 gcgctgattc aaaggttacc tcaacgcgac ccgccgctgc atcctttgtt gctcctcttg 2701 tcgcaaacaa acaaaaaacg tgctgtgctt tttcctttat cgtgtctttt tgcaaagtct 2761 agagtttacc gacaagacca gaatagcgct tatgtgtgtg tgtttgtgtg tgggtgtgtg 2821 tgcccatgtg tgacaaagag gccagtgctt tctctaaagc aagagcgcga ggttgctggc 2881 taaggactct aagtccctca cagtgatggc gtgttgcatt ttctaaaccg tcaatccaac 2941 acgtcggtag catatcggtt ttgtaggctc ttctggatac cttccccagt aagaccgcca 3001 ctgcttcgtg actgaagcac tgtaaatatc actgaaaccg tcgcgttgct ttgtctaatt 3061 atcaccgcct ttgccagcta tcgcctcagg ctgcgctctt tgactaatcc actcaccact 3121 tctctgcctt cttcctccgg ttgttgttgt gtcctgcgtg tacatggcgc gtgtcctttt 3181 cgagcaaaca gctgtctttt ctttcacgat aacacactca tattaaacgc gagtattact 3241 catcagtcaa cgtcacattc cgctctgtcc acttcgacct tacacctcta cttgtccaac 3301 tatcttccac ttgtcaagcg gctagcatgc ctttgtctca agaagaatcc accctcattg 3361 aaagagcaac ggctacaatc aacagcatcc ccatctctga agactacagc gtcgccagcg 3421 cagctctctc tagcgacggc cgcatcttca ctggtgtcaa tgtatatcat tttactgggg 3481 gaccttgtgc agaactcgtg gtgctgggca ctgctgctgc tgcggcagct ggcaacctga 3541 cttgtatcgt cgcgatcgga aatgagaaca ggggcatctt gagcccctgc ggacggtgcc 3601 gacaggtgct tctcgatctg catcctggga tcaaagccat agtgaaggac agtgatggac 3661 agccgacggc agttgggatt cgtgaattgc tgccctctgg ttatgtgtgg gagggctaac 3721 tcgagtgccc atttagttgg cttttccctt gtctcgtgtc ttttccgtgg aaaggttccc 3781 ggagtaatct gatggcacag cagggaggtg cgcctgcagg ttggttagga aggggggatg 3841 atgtaaaaga agaaaatggg gggattcgag ccccgcacag gtctctcaaa ttggatgcat 3901 agcttgagta ttctatagtg tcacctaaat agcttggcgt aatcatggtc atagctgttt 3961 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag 4021 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg 4081 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg 4141 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc 4201 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc 4261 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg 4321 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat 4381 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag 4441 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga 4501 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg 4561 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt 4621 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac 4681 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc 4741 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt 4801 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc 4861 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc 4921 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg 4981 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag 5041 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg 5101 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt 5161 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca 5221 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca 5281 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc 5341 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt 5401 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg 5461 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc 5521 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg 5581 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga 5641 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga 5701 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta 5761 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg 5821 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact 5881 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata 5941 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt 6001 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa 6061 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg atgcggtgtg aaataccgca 6121 cagatgcgta aggagaaaat accgcatcag gaaattgtaa gcgttaatat tttgttaaaa 6181 ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa 6241 atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac 6301 aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag 6361 ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt 6421 aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg 6481 gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca 6541 agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag 6601 ggcgcgtcca ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct 6661 cttcgctatt acgccagctg gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa 6721 cgccagggtt ttcccagtca cgacgttgta aaacgacggc cagtgaattg taatacgact 6781 cactata // LOCUS pPLOT-mNG-Neo-Neo 7591 bp ds-DNA circular 24-AUG-2016 DEFINITION pPLOT-mNG-Neo-Neo FEATURES Location/Qualifiers rep_origin complement(5136..5818) /label="ColE1 origin" /ApEinfo_revcolor=#b1ff67 /ApEinfo_fwdcolor=#b1ff67 CDS complement(5916..6575) /label="AmpR" /ApEinfo_revcolor=#ff9ccd /ApEinfo_fwdcolor=#ff9ccd misc_feature 2647..3722 /label="Cf PGKA/B" /ApEinfo_revcolor=#c7b0e3 /ApEinfo_fwdcolor=#c7b0e3 misc_feature 1777..1796 /label="Primer C-terminal For" /ApEinfo_revcolor=#ff9ccd /ApEinfo_fwdcolor=#ff9ccd misc_feature 3729..4523 /label="NeoR" /ApEinfo_revcolor=#b1ff67 /ApEinfo_fwdcolor=#b1ff67 CDS complement(7412..7480) /label="LacZ alpha" /ApEinfo_revcolor=#d59687 /ApEinfo_fwdcolor=#d59687 misc_feature 1813..2517 /label="mNeonGreen" /ApEinfo_revcolor=#b1ff67 /ApEinfo_fwdcolor=#b1ff67 misc_feature 1127..1683 /label="PGKB 5'" /ApEinfo_revcolor=#faac61 /ApEinfo_fwdcolor=#faac61 misc_feature 972..1120 /label="Ald3'UTR" /ApEinfo_revcolor=#b1ff67 /ApEinfo_fwdcolor=#b1ff67 primer_bind 7567..7591 /label="T7" /ApEinfo_revcolor=#f8d3a9 /ApEinfo_fwdcolor=#f8d3a9 3'UTR 4530..4678 /label="Ald3'UTR (short)(1)" /ApEinfo_revcolor=#85dae9 /ApEinfo_fwdcolor=#85dae9 primer_bind 7551..7568 /label="M13-fwd" /ApEinfo_revcolor=#75c6a9 /ApEinfo_fwdcolor=#75c6a9 misc_feature 1777..1806 /label="GS linker" /ApEinfo_revcolor=#84b0dc /ApEinfo_fwdcolor=#84b0dc primer_bind 16..35 /label="#161" /ApEinfo_revcolor=#b7e6d7 /ApEinfo_fwdcolor=#b7e6d7 primer_bind 1..3 /label="T7" /ApEinfo_revcolor=#85dae9 /ApEinfo_fwdcolor=#85dae9 CDS 1687..1776 /label="3xMyc" /ApEinfo_revcolor=#ffef86 /ApEinfo_fwdcolor=#ffef86 rep_origin complement(7088..7394) /label="F1 ori" /ApEinfo_revcolor=#b1ff67 /ApEinfo_fwdcolor=#b1ff67 misc_feature complement(2534..2553) /label="Primer N-terminal Rev" /ApEinfo_revcolor=#9eafd2 /ApEinfo_fwdcolor=#9eafd2 5'UTR 36..164 /label="Actin 5'" /ApEinfo_revcolor=#faac61 /ApEinfo_fwdcolor=#faac61 misc_binding complement(4774..4796) /label="LacO" /ApEinfo_revcolor=#ff9ccd /ApEinfo_fwdcolor=#ff9ccd misc_feature 171..965 /label="NeoR" /ApEinfo_revcolor=#d6b295 /ApEinfo_fwdcolor=#d6b295 CDS 2554..2643 /label="3xMyc" /ApEinfo_revcolor=#ffef86 /ApEinfo_fwdcolor=#ffef86 primer_bind complement(4679..4698) /label="#162" /ApEinfo_revcolor=#c7b0e3 /ApEinfo_fwdcolor=#c7b0e3 primer_bind complement(4717..4742) /label="SP6" /ApEinfo_revcolor=#d6b295 /ApEinfo_fwdcolor=#d6b295 misc_feature 2524..2553 /label="GS linker" /ApEinfo_revcolor=#d59687 /ApEinfo_fwdcolor=#d59687 primer_bind complement(4748..4768) /label="M13-rev" /ApEinfo_revcolor=#b4abac /ApEinfo_fwdcolor=#b4abac ORIGIN 1 GGGCGAATTG GGCCCgtata atgcagacct gctgcAAGCT AGGGGCACAG CAAGGTCTTC 61 TGAAATTCAT GTTTTTTTTT TTTTTACTCT GCATTGCAGT CTCCGCTCTT ATTTAGTTTT 121 GCTTTACGTA AGGTCTCGTT GCTGCCATAA AATAAGCTCT AGTGgaattc ATGATTGAAC 181 AAGATGGATT GCACGCAGGT TCTCCGGCCG CTTGGGTGGA GAGGCTATTC GGCTATGACT 241 GGGCACAACA GACAATCGGC TGCTCTGATG CCGCCGTGTT CCGGCTGTCA GCGCAGGGGC 301 GCCCGGTTCT TTTTGTCAAG ACCGACCTGT CCGGTGCCCT GAATGAACTG CAGGACGAGG 361 CAGCGCGGCT ATCGTGGCTG GCCACGACGG GCGTTCCTTG CGCAGCTGTG CTCGACGTTG 421 TCACTGAAGC GGGAAGGGAC TGGCTGCTAT TGGGCGAAGT GCCGGGGCAG GATCTCCTGT 481 CATCTCACCT TGCTCCTGCC GAGAAAGTAT CCATCATGGC TGATGCAATG CGGCGGCTGC 541 ATACGCTTGA TCCGGCTACC TGCCCATTCG ACCACCAAGC GAAACATCGC ATCGAGCGAG 601 CACGTACTCG GATGGAAGCC GGTCTTGTCG ATCAGGATGA TCTGGACGAA GAGCATCAGG 661 GGCTCGCGCC AGCCGAACTG TTCGCCAGGC TCAAGGCGCG CATGCCCGAC GGCGAGGATC 721 TCGTCGTGAC CCAcGGCGAT GCCTGCTTGC CGAATATCAT GGTGGAAAAT GGCCGCTTTT 781 CTGGATTCAT CGACTGTGGC CGGCTGGGTG TGGCGGACCG CTATCAGGAC ATAGCGTTGG 841 CTACCCGTGA TATTGCTGAA GAGCTTGGCG GCGAATGGGC TGACCGCTTC CTCGTGCTTT 901 ACGGTATCGC CGCTCCCGAT TCGCAGCGCA TCGCCTTCTA TCGCCTTCTT GACGAGTTCT 961 TCTGAccatg gtgcccattt agttggcttt tcccttgtct cgtgtctttt ccgtggaaag 1021 gttcccggag taatctgatg gcacagcagg gaggtgcgcc tgcaggttgg ttaggaaggg 1081 gggatgatgt aaaagaagaa aatgggggga ttcgagcccc acgcgttcga gaccgacaag 1141 accagaatag cgcttatgtg tgtgtgtttg tgtgtgggtg tgtgtgccca tgtgtgacaa 1201 agaggccagt gctttctcta aagcaagagc gcgaggttgc tggctaagga ctctaagtcc 1261 ctcacagtga tggcgtgttg cattttctaa accgtcaatc caacacgtcg gtagcatatc 1321 ggttttgtag gctcttctgg ataccttccc cagtaagacc gccactgctt cgtgactgaa 1381 gcactgtaaa tatcactgaa accgtcgcgt tgctttgtct aattatcacc gcctttgcca 1441 gctatcgcct caggctgcgc tctttgacta atccactcac cacttctctg ccttcttcct 1501 ccggttgttg ttgtgtcctg cgtgtacatg gcgcgtgtcc ttttcgagca aacagctgtc 1561 ttttctttca cgataacaca ctcatattaa acgcgagtat tactcatcag tcaacgtcac 1621 attccgctct gtccacttcg accttacacc tctacttgtc caactatctt ccacttgtca 1681 agcatggaac agaagctgat cagcgaagaa gacctggagc aaaagctcat tagcgaggag 1741 gacctcgagc agaagctgat tagcgaggag gacctgggtt ctggtagtgg ttccggttcc 1801 ggttctaagc ttgtctcgaa aggtgaggaa gacaatatgg ccagcttgcc agcaacgcac 1861 gagcttcata tatttgggtc gatcaacggt gtggatttcg acatggtggg tcaaggcaca 1921 ggtaatccta atgacgggta tgaagaactc aatttgaaga gcactaaggg agatctccag 1981 ttttccccgt ggattcttgt gccgcatatt ggttatggtt ttcatcagta cttaccatac 2041 cccgatggaa tgtcgccctt ccaagcagct atggtggacg gctcggggta tcaagtacat 2101 cgcaccatgc aatttgagga cggagcatcc ctcaccgtaa actaccgcta tacgtatgag 2161 gggagccaca taaaaggtga ggctcaagtt aaaggcacag gcttcccagc tgacggcccg 2221 gtcatgacca attcattaac cgctgctgat tggtgccgtt ctaagaagac ctaccctaac 2281 gacaagacga tcattagtac ttttaagtgg agttatacca caggaaacgg aaagcggtat 2341 cgaagcactg ctcgaacaac ctacacattt gcgaagccaa tggccgcgaa ctacttgaag 2401 aaccagccaa tgtatgtgtt tcgaaaaacg gaactgaagc attcgaaaac cgagcttaat 2461 tttaaagagt ggcagaaggc gtttacagac gtgatgggga tggacgaatt gtataaggga 2521 tccggatcag gatctggatc aggatcgggt agtgaacaga agctgatcag cgaagaagac 2581 ctggagcaaa agctcattag cgaggaggac ctcgagcaga agctgattag cgaggaggac 2641 ctgtaaccac caccaccact gagaattctg tattacgccg ttttaagagc tccttcgacg 2701 cctttttgtc cttcagctgc tccacggtga ctcgcttctc tctctttcca cagtgtctct 2761 tttttctttt cactctctat acaaatgtga gcgacctcct tttctgtaca acggccttcc 2821 ggcgtgtgct tttctcatcg acctctcctc gcttcttggg cactccttca tcgagcaaac 2881 aagacgaggg aggtgacgcg attggtgaga ccactacgag tatggacgga gctgtctgat 2941 accccctgtt ttttatcttg taccccgctc tgcagtgacg gtagcccgct gctgtgcttc 3001 ggtatcgccg cttcatacgc ttctctcttt tttcaacgtg cggcgctgat tcaaaggtta 3061 cctcaacgcg acccgccgct gcatcctttg ttgctcctct tgtcgcaaac aaacaaaaaa 3121 cgtgctgtgc tttttccttt atcgtgtctt tttgcaaagt ctagagttta ccgacaagac 3181 cagaatagcg cttatgtgtg tgtgtttgtg tgtgggtgtg tgtgcccatg tgtgacaaag 3241 aggccagtgc tttctctaaa gcaagagcgc gaggttgctg gctaaggact ctaagtccct 3301 cacagtgatg gcgtgttgca ttttctaaac cgtcaatcca acacgtcggt agcatatcgg 3361 ttttgtaggc tcttctggat accttcccca gtaagaccgc cactgcttcg tgactgaagc 3421 actgtaaata tcactgaaac cgtcgcgttg ctttgtctaa ttatcaccgc ctttgccagc 3481 tatcgcctca ggctgcgctc tttgactaat ccactcacca cttctctgcc ttcttcctcc 3541 ggttgttgtt gtgtcctgcg tgtacatggc gcgtgtcctt ttcgagcaaa cagctgtctt 3601 ttctttcacg ataacacact catattaaac gcgagtatta ctcatcagtc aacgtcacat 3661 tccgctctgt ccacttcgac cttacacctc tacttgtcca actatcttcc acttgtcaag 3721 cggctagcAT GATTGAACAA GATGGATTGC ACGCAGGTTC TCCGGCCGCT TGGGTGGAGA 3781 GGCTATTCGG CTATGACTGG GCACAACAGA CAATCGGCTG CTCTGATGCC GCCGTGTTCC 3841 GGCTGTCAGC GCAGGGGCGC CCGGTTCTTT TTGTCAAGAC CGACCTGTCC GGTGCCCTGA 3901 ATGAACTGCA GGACGAGGCA GCGCGGCTAT CGTGGCTGGC CACGACGGGC GTTCCTTGCG 3961 CAGCTGTGCT CGACGTTGTC ACTGAAGCGG GAAGGGACTG GCTGCTATTG GGCGAAGTGC 4021 CGGGGCAGGA TCTCCTGTCA TCTCACCTTG CTCCTGCCGA GAAAGTATCC ATCATGGCTG 4081 ATGCAATGCG GCGGCTGCAT ACGCTTGATC CGGCTACCTG CCCATTCGAC CACCAAGCGA 4141 AACATCGCAT CGAGCGAGCA CGTACTCGGA TGGAAGCCGG TCTTGTCGAT CAGGATGATC 4201 TGGACGAAGA GCATCAGGGG CTCGCGCCAG CCGAACTGTT CGCCAGGCTC AAGGCGCGCA 4261 TGCCCGACGG CGAGGATCTC GTCGTGACCC AcGGCGATGC CTGCTTGCCG AATATCATGG 4321 TGGAAAATGG CCGCTTTTCT GGATTCATCG ACTGTGGCCG GCTGGGTGTG GCGGACCGCT 4381 ATCAGGACAT AGCGTTGGCT ACCCGTGATA TTGCTGAAGA GCTTGGCGGC GAATGGGCTG 4441 ACCGCTTCCT CGTGCTTTAC GGTATCGCCG CTCCCGATTC GCAGCGCATC GCCTTCTATC 4501 GCCTTCTTGA CGAGTTCTTC TGActcgagT GCCCATTTAG TTGGCTTTTC CCTTGTCTCG 4561 TGTCTTTTCC GTGGAAAGGT TCCCGGAGTA ATCTGATGGC ACAGCAGGGA GGTGCGCCTG 4621 CAGGTTGGTT AGGAAGGGGG GATGATGTAA AAGAAGAAAA TGGGGGGATT CGAGCCCCGC 4681 ACAGGTCTCT CAAATTGGAT GCATAGCTTG AGTATTCTAT AGTGTCACCT AAATAGCTTG 4741 GCGTAATCAT GGTCATAGCT GTTTCCTGTG TGAAATTGTT ATCCGCTCAC AATTCCACAC 4801 AACATACGAG CCGGAAGCAT AAAGTGTAAA GCCTGGGGTG CCTAATGAGT GAGCTAACTC 4861 ACATTAATTG CGTTGCGCTC ACTGCCCGCT TTCCAGTCGG GAAACCTGTC GTGCCAGCTG 4921 CATTAATGAA TCGGCCAACG CGCGGGGAGA GGCGGTTTGC GTATTGGGCG CTCTTCCGCT 4981 TCCTCGCTCA CTGACTCGCT GCGCTCGGTC GTTCGGCTGC GGCGAGCGGT ATCAGCTCAC 5041 TCAAAGGCGG TAATACGGTT ATCCACAGAA TCAGGGGATA ACGCAGGAAA GAACATGTGA 5101 GCAAAAGGCC AGCAAAAGGC CAGGAACCGT AAAAAGGCCG CGTTGCTGGC GTTTTTCCAT 5161 AGGCTCCGCC CCCCTGACGA GCATCACAAA AATCGACGCT CAAGTCAGAG GTGGCGAAAC 5221 CCGACAGGAC TATAAAGATA CCAGGCGTTT CCCCCTGGAA GCTCCCTCGT GCGCTCTCCT 5281 GTTCCGACCC TGCCGCTTAC CGGATACCTG TCCGCCTTTC TCCCTTCGGG AAGCGTGGCG 5341 CTTTCTCATA GCTCACGCTG TAGGTATCTC AGTTCGGTGT AGGTCGTTCG CTCCAAGCTG 5401 GGCTGTGTGC ACGAACCCCC CGTTCAGCCC GACCGCTGCG CCTTATCCGG TAACTATCGT 5461 CTTGAGTCCA ACCCGGTAAG ACACGACTTA TCGCCACTGG CAGCAGCCAC TGGTAACAGG 5521 ATTAGCAGAG CGAGGTATGT AGGCGGTGCT ACAGAGTTCT TGAAGTGGTG GCCTAACTAC 5581 GGCTACACTA GAAGAACAGT ATTTGGTATC TGCGCTCTGC TGAAGCCAGT TACCTTCGGA 5641 AAAAGAGTTG GTAGCTCTTG ATCCGGCAAA CAAACCACCG CTGGTAGCGG TGGTTTTTTT 5701 GTTTGCAAGC AGCAGATTAC GCGCAGAAAA AAAGGATCTC AAGAAGATCC TTTGATCTTT 5761 TCTACGGGGT CTGACGCTCA GTGGAACGAA AACTCACGTT AAGGGATTTT GGTCATGAGA 5821 TTATCAAAAA GGATCTTCAC CTAGATCCTT TTAAATTAAA AATGAAGTTT TAAATCAATC 5881 TAAAGTATAT ATGAGTAAAC TTGGTCTGAC AGTTACCAAT GCTTAATCAG TGAGGCACCT 5941 ATCTCAGCGA TCTGTCTATT TCGTTCATCC ATAGTTGCCT GACTCCCCGT CGTGTAGATA 6001 ACTACGATAC GGGAGGGCTT ACCATCTGGC CCCAGTGCTG CAATGATACC GCGAGACCCA 6061 CGCTCACCGG CTCCAGATTT ATCAGCAATA AACCAGCCAG CCGGAAGGGC CGAGCGCAGA 6121 AGTGGTCCTG CAACTTTATC CGCCTCCATC CAGTCTATTA ATTGTTGCCG GGAAGCTAGA 6181 GTAAGTAGTT CGCCAGTTAA TAGTTTGCGC AACGTTGTTG CCATTGCTAC AGGCATCGTG 6241 GTGTCACGCT CGTCGTTTGG TATGGCTTCA TTCAGCTCCG GTTCCCAACG ATCAAGGCGA 6301 GTTACATGAT CCCCCATGTT GTGCAAAAAA GCGGTTAGCT CCTTCGGTCC TCCGATCGTT 6361 GTCAGAAGTA AGTTGGCCGC AGTGTTATCA CTCATGGTTA TGGCAGCACT GCATAATTCT 6421 CTTACTGTCA TGCCATCCGT AAGATGCTTT TCTGTGACTG GTGAGTACTC AACCAAGTCA 6481 TTCTGAGAAT AGTGTATGCG GCGACCGAGT TGCTCTTGCC CGGCGTCAAT ACGGGATAAT 6541 ACCGCGCCAC ATAGCAGAAC TTTAAAAGTG CTCATCATTG GAAAACGTTC TTCGGGGCGA 6601 AAACTCTCAA GGATCTTACC GCTGTTGAGA TCCAGTTCGA TGTAACCCAC TCGTGCACCC 6661 AACTGATCTT CAGCATCTTT TACTTTCACC AGCGTTTCTG GGTGAGCAAA AACAGGAAGG 6721 CAAAATGCCG CAAAAAAGGG AATAAGGGCG ACACGGAAAT GTTGAATACT CATACTCTTC 6781 CTTTTTCAAT ATTATTGAAG CATTTATCAG GGTTATTGTC TCATGAGCGG ATACATATTT 6841 GAATGTATTT AGAAAAATAA ACAAATAGGG GTTCCGCGCA CATTTCCCCG AAAAGTGCCA 6901 CCTGATGCGG TGTGAAATAC CGCACAGATG CGTAAGGAGA AAATACCGCA TCAGGAAATT 6961 GTAAGCGTTA ATATTTTGTT AAAATTCGCG TTAAATTTTT GTTAAATCAG CTCATTTTTT 7021 AACCAATAGG CCGAAATCGG CAAAATCCCT TATAAATCAA AAGAATAGAC CGAGATAGGG 7081 TTGAGTGTTG TTCCAGTTTG GAACAAGAGT CCACTATTAA AGAACGTGGA CTCCAACGTC 7141 AAAGGGCGAA AAACCGTCTA TCAGGGCGAT GGCCCACTAC GTGAACCATC ACCCTAATCA 7201 AGTTTTTTGG GGTCGAGGTG CCGTAAAGCA CTAAATCGGA ACCCTAAAGG GAGCCCCCGA 7261 TTTAGAGCTT GACGGGGAAA GCCGGCGAAC GTGGCGAGAA AGGAAGGGAA GAAAGCGAAA 7321 GGAGCGGGCG CTAGGGCGCT GGCAAGTGTA GCGGTCACGC TGCGCGTAAC CACCACACCC 7381 GCCGCGCTTA ATGCGCCGCT ACAGGGCGCG TCCATTCGCC ATTCAGGCTG CGCAACTGTT 7441 GGGAAGGGCG ATCGGTGCGG GCCTCTTCGC TATTACGCCA GCTGGCGAAA GGGGGATGTG 7501 CTGCAAGGCG ATTAAGTTGG GTAACGCCAG GGTTTTCCCA GTCACGACGT TGTAAAACGA 7561 CGGCCAGTGA ATTGTAATAC GACTCACTAT A // LOCUS pPLOT-mNG-Puro-Puro 7201 bp ds-DNA circular 27-MAY-2016 DEFINITION pPLOT-mNG-Puro-Puro FEATURES Location/Qualifiers primer_bind 7161..7178 /label="M13-fwd" /ApEinfo_revcolor=#ffef86 /ApEinfo_fwdcolor=#ffef86 primer_bind 1..3 /label="T7" /ApEinfo_revcolor=#d59687 /ApEinfo_fwdcolor=#d59687 misc_feature 2452..3527 /label="Cf PGKA/B" /ApEinfo_revcolor=#c7b0e3 /ApEinfo_fwdcolor=#c7b0e3 primer_bind 7177..3 /label="T7" /ApEinfo_revcolor=#85dae9 /ApEinfo_fwdcolor=#85dae9 CDS complement(5526..6185) /label="AmpR" /ApEinfo_revcolor=#d6b295 /ApEinfo_fwdcolor=#d6b295 primer_bind 16..35 /label="#161" /ApEinfo_revcolor=#f58a5e /ApEinfo_fwdcolor=#f58a5e primer_bind complement(4289..4308) /label="#162" /ApEinfo_revcolor=#f58a5e /ApEinfo_fwdcolor=#f58a5e misc_feature 3534..4133 /label="PuroR" /ApEinfo_revcolor=#d59687 /ApEinfo_fwdcolor=#d59687 5'UTR 36..164 /label="Actin 5'" /ApEinfo_revcolor=#b7e6d7 /ApEinfo_fwdcolor=#b7e6d7 misc_feature 777..925 /label="Ald3'UTR" /ApEinfo_revcolor=#b1ff67 /ApEinfo_fwdcolor=#b1ff67 misc_feature 1618..2322 /label="mNeonGreen" /ApEinfo_revcolor=#b1ff67 /ApEinfo_fwdcolor=#b1ff67 misc_binding complement(4384..4406) /label="LacO" /ApEinfo_revcolor=#f8d3a9 /ApEinfo_fwdcolor=#f8d3a9 primer_bind complement(4327..4352) /label="SP6" /ApEinfo_revcolor=#84b0dc /ApEinfo_fwdcolor=#84b0dc primer_bind complement(4358..4378) /label="M13-rev" /ApEinfo_revcolor=#b7e6d7 /ApEinfo_fwdcolor=#b7e6d7 misc_feature 171..770 /label="PuroR" /ApEinfo_revcolor=#d59687 /ApEinfo_fwdcolor=#d59687 misc_feature complement(2339..2358) /label="Primer N-terminal Rev" /ApEinfo_revcolor=#9eafd2 /ApEinfo_fwdcolor=#9eafd2 CDS 2359..2448 /label="3xMyc" /ApEinfo_revcolor=#ffef86 /ApEinfo_fwdcolor=#ffef86 CDS complement(7022..7090) /label="LacZ alpha" /ApEinfo_revcolor=#b4abac /ApEinfo_fwdcolor=#b4abac primer_bind 7177..7201 /label="T7" /ApEinfo_revcolor=#b7e6d7 /ApEinfo_fwdcolor=#b7e6d7 misc_feature 1582..1611 /label="GS linker" /ApEinfo_revcolor=#84b0dc /ApEinfo_fwdcolor=#84b0dc misc_feature 932..1488 /label="PGKB 5'" /ApEinfo_revcolor=#faac61 /ApEinfo_fwdcolor=#faac61 CDS 1492..1581 /label="3xMyc" /ApEinfo_revcolor=#ffef86 /ApEinfo_fwdcolor=#ffef86 misc_feature 1582..1601 /label="Primer C-terminal For" /ApEinfo_revcolor=#ff9ccd /ApEinfo_fwdcolor=#ff9ccd rep_origin complement(4746..5428) /label="ColE1 origin" /ApEinfo_revcolor=#ff9ccd /ApEinfo_fwdcolor=#ff9ccd rep_origin complement(6698..7004) /label="F1 ori" /ApEinfo_revcolor=#75c6a9 /ApEinfo_fwdcolor=#75c6a9 misc_feature 2329..2358 /label="GS linker" /ApEinfo_revcolor=#d59687 /ApEinfo_fwdcolor=#d59687 3'UTR 4140..4288 /label="Ald3'UTR (short)(1)" /ApEinfo_revcolor=#c7b0e3 /ApEinfo_fwdcolor=#c7b0e3 ORIGIN 1 GGGCGAATTG GGCCCgtata atgcagacct gctgcAAGCT AGGGGCACAG CAAGGTCTTC 61 TGAAATTCAT GTTTTTTTTT TTTTTACTCT GCATTGCAGT CTCCGCTCTT ATTTAGTTTT 121 GCTTTACGTA AGGTCTCGTT GCTGCCATAA AATAAGCTCT AGTGgaattc ATGACTGAAT 181 ACAAGCCAAC GGTTCGTCTT GCTACGCGAG ACGATGTGCC GCGGGCCGTA CGGACGCTGG 241 CTGCTGCGTT CGCTGATTAT CCCGCCACCC GACATACGGT TGACCCAGAT CGACACATTG 301 AGCGAGTCAC AGAACTTCAA GAGTTATTCT TAACACGGGT GGGCTTGGAC ATTGGTAAGG 361 TTTGGGTTGC GGACGATGGC GCGGCTGTTG CGGTGTGGAC GACGCCAGAG TCAGTCGAAG 421 CGGGCGCAGT GTTTGCTGAG ATCGGTCCCC GAATGGCGGA GCTTAGCGGG TCACGCCTCG 481 CTGCACAACA GCAGATGGAA GGTCTGTTGG CACCACATAG ACCTAAGGAG CCGGCTTGGT 541 TCTTAGCGAC AGTTGGCGTC AGTCCGGACC ACCAAGGTAA GGGGCTGGGT TCAGCGGTTG 601 TTCTTCCAGG AGTAGAGGCT GCGGAAAGGG CAGGTGTGCC GGCGTTCCTT GAAACGTCTG 661 CACCACGTAA CCTTCCTTTT TACGAGAGAT TGGGGTTTAC AGTAACAGCA GATGTTGAAG 721 TGCCTGAAGG GCCGAGGACA TGGTGCATGA CACGTAAGCC GGGAGCCTAA ccatggtgcc 781 catttagttg gcttttccct tgtctcgtgt cttttccgtg gaaaggttcc cggagtaatc 841 tgatggcaca gcagggaggt gcgcctgcag gttggttagg aaggggggat gatgtaaaag 901 aagaaaatgg ggggattcga gccccacgcg ttcgagaccg acaagaccag aatagcgctt 961 atgtgtgtgt gtttgtgtgt gggtgtgtgt gcccatgtgt gacaaagagg ccagtgcttt 1021 ctctaaagca agagcgcgag gttgctggct aaggactcta agtccctcac agtgatggcg 1081 tgttgcattt tctaaaccgt caatccaaca cgtcggtagc atatcggttt tgtaggctct 1141 tctggatacc ttccccagta agaccgccac tgcttcgtga ctgaagcact gtaaatatca 1201 ctgaaaccgt cgcgttgctt tgtctaatta tcaccgcctt tgccagctat cgcctcaggc 1261 tgcgctcttt gactaatcca ctcaccactt ctctgccttc ttcctccggt tgttgttgtg 1321 tcctgcgtgt acatggcgcg tgtccttttc gagcaaacag ctgtcttttc tttcacgata 1381 acacactcat attaaacgcg agtattactc atcagtcaac gtcacattcc gctctgtcca 1441 cttcgacctt acacctctac ttgtccaact atcttccact tgtcaagcat ggaacagaag 1501 ctgatcagcg aagaagacct ggagcaaaag ctcattagcg aggaggacct cgagcagaag 1561 ctgattagcg aggaggacct gggttctggt agtggttccg gttccggttc taagcttgtc 1621 tcgaaaggtg aggaagacaa tatggccagc ttgccagcaa cgcacgagct tcatatattt 1681 gggtcgatca acggtgtgga tttcgacatg gtgggtcaag gcacaggtaa tcctaatgac 1741 gggtatgaag aactcaattt gaagagcact aagggagatc tccagttttc cccgtggatt 1801 cttgtgccgc atattggtta tggttttcat cagtacttac cataccccga tggaatgtcg 1861 cccttccaag cagctatggt ggacggctcg gggtatcaag tacatcgcac catgcaattt 1921 gaggacggag catccctcac cgtaaactac cgctatacgt atgaggggag ccacataaaa 1981 ggtgaggctc aagttaaagg cacaggcttc ccagctgacg gcccggtcat gaccaattca 2041 ttaaccgctg ctgattggtg ccgttctaag aagacctacc ctaacgacaa gacgatcatt 2101 agtactttta agtggagtta taccacagga aacggaaagc ggtatcgaag cactgctcga 2161 acaacctaca catttgcgaa gccaatggcc gcgaactact tgaagaacca gccaatgtat 2221 gtgtttcgaa aaacggaact gaagcattcg aaaaccgagc ttaattttaa agagtggcag 2281 aaggcgttta cagacgtgat ggggatggac gaattgtata agggatccgg atcaggatct 2341 ggatcaggat cgggtagtga acagaagctg atcagcgaag aagacctgga gcaaaagctc 2401 attagcgagg aggacctcga gcagaagctg attagcgagg aggacctgta accaccacca 2461 ccactgagaa ttctgtatta cgccgtttta agagctcctt cgacgccttt ttgtccttca 2521 gctgctccac ggtgactcgc ttctctctct ttccacagtg tctctttttt cttttcactc 2581 tctatacaaa tgtgagcgac ctccttttct gtacaacggc cttccggcgt gtgcttttct 2641 catcgacctc tcctcgcttc ttgggcactc cttcatcgag caaacaagac gagggaggtg 2701 acgcgattgg tgagaccact acgagtatgg acggagctgt ctgatacccc ctgtttttta 2761 tcttgtaccc cgctctgcag tgacggtagc ccgctgctgt gcttcggtat cgccgcttca 2821 tacgcttctc tcttttttca acgtgcggcg ctgattcaaa ggttacctca acgcgacccg 2881 ccgctgcatc ctttgttgct cctcttgtcg caaacaaaca aaaaacgtgc tgtgcttttt 2941 cctttatcgt gtctttttgc aaagtctaga gtttaccgac aagaccagaa tagcgcttat 3001 gtgtgtgtgt ttgtgtgtgg gtgtgtgtgc ccatgtgtga caaagaggcc agtgctttct 3061 ctaaagcaag agcgcgaggt tgctggctaa ggactctaag tccctcacag tgatggcgtg 3121 ttgcattttc taaaccgtca atccaacacg tcggtagcat atcggttttg taggctcttc 3181 tggatacctt ccccagtaag accgccactg cttcgtgact gaagcactgt aaatatcact 3241 gaaaccgtcg cgttgctttg tctaattatc accgcctttg ccagctatcg cctcaggctg 3301 cgctctttga ctaatccact caccacttct ctgccttctt cctccggttg ttgttgtgtc 3361 ctgcgtgtac atggcgcgtg tccttttcga gcaaacagct gtcttttctt tcacgataac 3421 acactcatat taaacgcgag tattactcat cagtcaacgt cacattccgc tctgtccact 3481 tcgaccttac acctctactt gtccaactat cttccacttg tcaagcggct agcATGACTG 3541 AATACAAGCC AACGGTTCGT CTTGCTACGC GAGACGATGT GCCGCGGGCC GTACGGACGC 3601 TGGCTGCTGC GTTCGCTGAT TATCCCGCCA CCCGACATAC GGTTGACCCA GATCGACACA 3661 TTGAGCGAGT CACAGAACTT CAAGAGTTAT TCTTAACACG GGTGGGCTTG GACATTGGTA 3721 AGGTTTGGGT TGCGGACGAT GGCGCGGCTG TTGCGGTGTG GACGACGCCA GAGTCAGTCG 3781 AAGCGGGCGC AGTGTTTGCT GAGATCGGTC CCCGAATGGC GGAGCTTAGC GGGTCACGCC 3841 TCGCTGCACA ACAGCAGATG GAAGGTCTGT TGGCACCACA TAGACCTAAG GAGCCGGCTT 3901 GGTTCTTAGC GACAGTTGGC GTCAGTCCGG ACCACCAAGG TAAGGGGCTG GGTTCAGCGG 3961 TTGTTCTTCC AGGAGTAGAG GCTGCGGAAA GGGCAGGTGT GCCGGCGTTC CTTGAAACGT 4021 CTGCACCACG TAACCTTCCT TTTTACGAGA GATTGGGGTT TACAGTAACA GCAGATGTTG 4081 AAGTGCCTGA AGGGCCGAGG ACATGGTGCA TGACACGTAA GCCGGGAGCC TAActcgagT 4141 GCCCATTTAG TTGGCTTTTC CCTTGTCTCG TGTCTTTTCC GTGGAAAGGT TCCCGGAGTA 4201 ATCTGATGGC ACAGCAGGGA GGTGCGCCTG CAGGTTGGTT AGGAAGGGGG GATGATGTAA 4261 AAGAAGAAAA TGGGGGGATT CGAGCCCCGC ACAGGTCTCT CAAATTGGAT GCATAGCTTG 4321 AGTATTCTAT AGTGTCACCT AAATAGCTTG GCGTAATCAT GGTCATAGCT GTTTCCTGTG 4381 TGAAATTGTT ATCCGCTCAC AATTCCACAC AACATACGAG CCGGAAGCAT AAAGTGTAAA 4441 GCCTGGGGTG CCTAATGAGT GAGCTAACTC ACATTAATTG CGTTGCGCTC ACTGCCCGCT 4501 TTCCAGTCGG GAAACCTGTC GTGCCAGCTG CATTAATGAA TCGGCCAACG CGCGGGGAGA 4561 GGCGGTTTGC GTATTGGGCG CTCTTCCGCT TCCTCGCTCA CTGACTCGCT GCGCTCGGTC 4621 GTTCGGCTGC GGCGAGCGGT ATCAGCTCAC TCAAAGGCGG TAATACGGTT ATCCACAGAA 4681 TCAGGGGATA ACGCAGGAAA GAACATGTGA GCAAAAGGCC AGCAAAAGGC CAGGAACCGT 4741 AAAAAGGCCG CGTTGCTGGC GTTTTTCCAT AGGCTCCGCC CCCCTGACGA GCATCACAAA 4801 AATCGACGCT CAAGTCAGAG GTGGCGAAAC CCGACAGGAC TATAAAGATA CCAGGCGTTT 4861 CCCCCTGGAA GCTCCCTCGT GCGCTCTCCT GTTCCGACCC TGCCGCTTAC CGGATACCTG 4921 TCCGCCTTTC TCCCTTCGGG AAGCGTGGCG CTTTCTCATA GCTCACGCTG TAGGTATCTC 4981 AGTTCGGTGT AGGTCGTTCG CTCCAAGCTG GGCTGTGTGC ACGAACCCCC CGTTCAGCCC 5041 GACCGCTGCG CCTTATCCGG TAACTATCGT CTTGAGTCCA ACCCGGTAAG ACACGACTTA 5101 TCGCCACTGG CAGCAGCCAC TGGTAACAGG ATTAGCAGAG CGAGGTATGT AGGCGGTGCT 5161 ACAGAGTTCT TGAAGTGGTG GCCTAACTAC GGCTACACTA GAAGAACAGT ATTTGGTATC 5221 TGCGCTCTGC TGAAGCCAGT TACCTTCGGA AAAAGAGTTG GTAGCTCTTG ATCCGGCAAA 5281 CAAACCACCG CTGGTAGCGG TGGTTTTTTT GTTTGCAAGC AGCAGATTAC GCGCAGAAAA 5341 AAAGGATCTC AAGAAGATCC TTTGATCTTT TCTACGGGGT CTGACGCTCA GTGGAACGAA 5401 AACTCACGTT AAGGGATTTT GGTCATGAGA TTATCAAAAA GGATCTTCAC CTAGATCCTT 5461 TTAAATTAAA AATGAAGTTT TAAATCAATC TAAAGTATAT ATGAGTAAAC TTGGTCTGAC 5521 AGTTACCAAT GCTTAATCAG TGAGGCACCT ATCTCAGCGA TCTGTCTATT TCGTTCATCC 5581 ATAGTTGCCT GACTCCCCGT CGTGTAGATA ACTACGATAC GGGAGGGCTT ACCATCTGGC 5641 CCCAGTGCTG CAATGATACC GCGAGACCCA CGCTCACCGG CTCCAGATTT ATCAGCAATA 5701 AACCAGCCAG CCGGAAGGGC CGAGCGCAGA AGTGGTCCTG CAACTTTATC CGCCTCCATC 5761 CAGTCTATTA ATTGTTGCCG GGAAGCTAGA GTAAGTAGTT CGCCAGTTAA TAGTTTGCGC 5821 AACGTTGTTG CCATTGCTAC AGGCATCGTG GTGTCACGCT CGTCGTTTGG TATGGCTTCA 5881 TTCAGCTCCG GTTCCCAACG ATCAAGGCGA GTTACATGAT CCCCCATGTT GTGCAAAAAA 5941 GCGGTTAGCT CCTTCGGTCC TCCGATCGTT GTCAGAAGTA AGTTGGCCGC AGTGTTATCA 6001 CTCATGGTTA TGGCAGCACT GCATAATTCT CTTACTGTCA TGCCATCCGT AAGATGCTTT 6061 TCTGTGACTG GTGAGTACTC AACCAAGTCA TTCTGAGAAT AGTGTATGCG GCGACCGAGT 6121 TGCTCTTGCC CGGCGTCAAT ACGGGATAAT ACCGCGCCAC ATAGCAGAAC TTTAAAAGTG 6181 CTCATCATTG GAAAACGTTC TTCGGGGCGA AAACTCTCAA GGATCTTACC GCTGTTGAGA 6241 TCCAGTTCGA TGTAACCCAC TCGTGCACCC AACTGATCTT CAGCATCTTT TACTTTCACC 6301 AGCGTTTCTG GGTGAGCAAA AACAGGAAGG CAAAATGCCG CAAAAAAGGG AATAAGGGCG 6361 ACACGGAAAT GTTGAATACT CATACTCTTC CTTTTTCAAT ATTATTGAAG CATTTATCAG 6421 GGTTATTGTC TCATGAGCGG ATACATATTT GAATGTATTT AGAAAAATAA ACAAATAGGG 6481 GTTCCGCGCA CATTTCCCCG AAAAGTGCCA CCTGATGCGG TGTGAAATAC CGCACAGATG 6541 CGTAAGGAGA AAATACCGCA TCAGGAAATT GTAAGCGTTA ATATTTTGTT AAAATTCGCG 6601 TTAAATTTTT GTTAAATCAG CTCATTTTTT AACCAATAGG CCGAAATCGG CAAAATCCCT 6661 TATAAATCAA AAGAATAGAC CGAGATAGGG TTGAGTGTTG TTCCAGTTTG GAACAAGAGT 6721 CCACTATTAA AGAACGTGGA CTCCAACGTC AAAGGGCGAA AAACCGTCTA TCAGGGCGAT 6781 GGCCCACTAC GTGAACCATC ACCCTAATCA AGTTTTTTGG GGTCGAGGTG CCGTAAAGCA 6841 CTAAATCGGA ACCCTAAAGG GAGCCCCCGA TTTAGAGCTT GACGGGGAAA GCCGGCGAAC 6901 GTGGCGAGAA AGGAAGGGAA GAAAGCGAAA GGAGCGGGCG CTAGGGCGCT GGCAAGTGTA 6961 GCGGTCACGC TGCGCGTAAC CACCACACCC GCCGCGCTTA ATGCGCCGCT ACAGGGCGCG 7021 TCCATTCGCC ATTCAGGCTG CGCAACTGTT GGGAAGGGCG ATCGGTGCGG GCCTCTTCGC 7081 TATTACGCCA GCTGGCGAAA GGGGGATGTG CTGCAAGGCG ATTAAGTTGG GTAACGCCAG 7141 GGTTTTCCCA GTCACGACGT TGTAAAACGA CGGCCAGTGA ATTGTAATAC GACTCACTAT 7201 A // LOCUS pPLOT-nanoLuc-Phleo-Phleo 6571 bp ds-DNA circular SYN 04-NOV-2016 DEFINITION pPLOT-nanoLuc-Phleo-Phleo ACCESSION . VERSION . KEYWORDS pPLOT-nanoLuc-Phleo-Phle SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6571) AUTHORS . TITLE Direct Submission JOURNAL Exported Friday, Nov 4, 2016 from SnapGene Viewer 3.2.1 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6571 /organism="synthetic DNA construct" /mol_type="other DNA" misc_feature 16..35 /note="#161" misc_feature 36..164 /note="Actin 5'" CDS 171..551 /codon_start=1 /note="BleoR" /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR EFALRDPAGNCVHFVAEEQDPW" misc_feature 558..706 /note="Ald3'UTR" misc_feature 713..1269 /note="PGKB 5'" CDS 1273..1362 /codon_start=1 /product="3 tandem Myc epitope tags" /note="3xMyc" /translation="EQKLISEEDLEQKLISEEDLEQKLISEEDL" misc_feature 1363..1392 /note="GS linker" misc_feature 1399..1911 /note="nanoLuc" misc_feature 1918..1947 /note="GS linker" CDS 1948..2037 /codon_start=1 /product="3 tandem Myc epitope tags" /note="3xMyc" /translation="EQKLISEEDLEQKLISEEDLEQKLISEEDL" misc_feature 2041..3116 /note="Cf PGKA/B" CDS 3123..3503 /codon_start=1 /note="BleoR" /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR EFALRDPAGNCVHFVAEEQDPW" misc_feature 3510..3658 /note="Ald3'UTR" promoter complement(3696..3714) /note="SP6 promoter" /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind complement(3732..3748) /note="M13 rev" /note="common sequencing primer, one of multiple similar variants" protein_bind 3756..3772 /bound_moiety="lac repressor encoded by lacI" /note="lac operator" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." promoter complement(3780..3810) /note="lac promoter" /note="promoter for the E. coli lac operon" protein_bind 3825..3846 /bound_moiety="E. coli catabolite activator protein" /note="CAP binding site" /note="CAP binding activates transcription in the presence of cAMP." rep_origin complement(4134..4722) /direction=LEFT /note="ori" /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(4893..5753) /codon_start=1 /gene="bla" /product="beta-lactamase" /note="AmpR" /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" promoter complement(5754..5858) /gene="bla" /note="AmpR promoter" rep_origin complement(5936..6391) /direction=LEFT /note="f1 ori" /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind 6532..6548 /note="M13 fwd" /note="common sequencing primer, one of multiple similar variants" promoter 6555..2 /note="T7 promoter" /note="promoter for bacteriophage T7 RNA polymerase" ORIGIN 1 gggcgaattg ggcccgtata atgcagacct gctgcaagct aggggcacag caaggtcttc 61 tgaaattcat gttttttttt tttttactct gcattgcagt ctccgctctt atttagtttt 121 gctttacgta aggtctcgtt gctgccataa aataagctct agtggaattc atggctaaac 181 tcacttctgc tgtaccagta ctcactgcta gagatgtagc tggagctgta gagttctgga 241 ctgatagact cggattctct agagatttcg tagaggatga tttcgctgga gtagtaagag 301 atgatgtaac tctcttcata tctgctgtac aagatcaagt agtaccagat aacactctcg 361 cttgggtatg ggtaagagga ctcgatgagc tctacgctga gtggtctgag gtagtatcta 421 ctaacttcag agatgcttct ggaccagcta tgactgagat aggagagcaa ccgtggggaa 481 gagagttcgc tctcagagat ccagctggaa actgcgtaca cttcgtagct gaggagcaag 541 atccatggta accatggtgc ccatttagtt ggcttttccc ttgtctcgtg tcttttccgt 601 ggaaaggttc ccggagtaat ctgatggcac agcagggagg tgcgcctgca ggttggttag 661 gaagggggga tgatgtaaaa gaagaaaatg gggggattcg agccccacgc gttcgagacc 721 gacaagacca gaatagcgct tatgtgtgtg tgtttgtgtg tgggtgtgtg tgcccatgtg 781 tgacaaagag gccagtgctt tctctaaagc aagagcgcga ggttgctggc taaggactct 841 aagtccctca cagtgatggc gtgttgcatt ttctaaaccg tcaatccaac acgtcggtag 901 catatcggtt ttgtaggctc ttctggatac cttccccagt aagaccgcca ctgcttcgtg 961 actgaagcac tgtaaatatc actgaaaccg tcgcgttgct ttgtctaatt atcaccgcct 1021 ttgccagcta tcgcctcagg ctgcgctctt tgactaatcc actcaccact tctctgcctt 1081 cttcctccgg ttgttgttgt gtcctgcgtg tacatggcgc gtgtcctttt cgagcaaaca 1141 gctgtctttt ctttcacgat aacacactca tattaaacgc gagtattact catcagtcaa 1201 cgtcacattc cgctctgtcc acttcgacct tacacctcta cttgtccaac tatcttccac 1261 ttgtcaagca tggaacagaa gctgatcagc gaagaagacc tggagcaaaa gctcattagc 1321 gaggaggacc tcgagcagaa gctgattagc gaggaggacc tgggttctgg tagtggttcc 1381 ggttccggtt ctaagcttat ggtatttact ttggaggatt ttgtagggga ttggcgccag 1441 acggccggct acaatctgga tcaggtactg gagcaaggcg gtgtttcgtc gttgtttcag 1501 aacctggggg tgtcagtaac ccctattcag aggatagtcc tttccggtga gaatggcctt 1561 aaaattgata tccacgtcat aattccttat gaagggttga gcggggacca aatggggcag 1621 atagagaaga ttttcaaggt agtttaccca gtcgatgacc atcacttcaa agttatattg 1681 cattatggga ctctggtcat cgacggtgtc acacctaata tgattgatta cttcggccgc 1741 ccatacgaag gaatagctgt gtttgatggg aagaagataa ctgtgacggg aactctgtgg 1801 aatggtaaca aaataatcga cgagaggctt ataaatcccg atggaagcct cctgttccgt 1861 gtaacgatca acggcgtaac tggttggcga ctgtgtgagc ggatacttgc aggatccgga 1921 tcaggatctg gatcaggatc gggtagtgaa cagaagctga tcagcgaaga agacctggag 1981 caaaagctca ttagcgagga ggacctcgag cagaagctga ttagcgagga ggacctgtaa 2041 ccaccaccac cactgagaat tctgtattac gccgttttaa gagctccttc gacgcctttt 2101 tgtccttcag ctgctccacg gtgactcgct tctctctctt tccacagtgt ctcttttttc 2161 ttttcactct ctatacaaat gtgagcgacc tccttttctg tacaacggcc ttccggcgtg 2221 tgcttttctc atcgacctct cctcgcttct tgggcactcc ttcatcgagc aaacaagacg 2281 agggaggtga cgcgattggt gagaccacta cgagtatgga cggagctgtc tgataccccc 2341 tgttttttat cttgtacccc gctctgcagt gacggtagcc cgctgctgtg cttcggtatc 2401 gccgcttcat acgcttctct cttttttcaa cgtgcggcgc tgattcaaag gttacctcaa 2461 cgcgacccgc cgctgcatcc tttgttgctc ctcttgtcgc aaacaaacaa aaaacgtgct 2521 gtgctttttc ctttatcgtg tctttttgca aagtctagag tttaccgaca agaccagaat 2581 agcgcttatg tgtgtgtgtt tgtgtgtggg tgtgtgtgcc catgtgtgac aaagaggcca 2641 gtgctttctc taaagcaaga gcgcgaggtt gctggctaag gactctaagt ccctcacagt 2701 gatggcgtgt tgcattttct aaaccgtcaa tccaacacgt cggtagcata tcggttttgt 2761 aggctcttct ggataccttc cccagtaaga ccgccactgc ttcgtgactg aagcactgta 2821 aatatcactg aaaccgtcgc gttgctttgt ctaattatca ccgcctttgc cagctatcgc 2881 ctcaggctgc gctctttgac taatccactc accacttctc tgccttcttc ctccggttgt 2941 tgttgtgtcc tgcgtgtaca tggcgcgtgt ccttttcgag caaacagctg tcttttcttt 3001 cacgataaca cactcatatt aaacgcgagt attactcatc agtcaacgtc acattccgct 3061 ctgtccactt cgaccttaca cctctacttg tccaactatc ttccacttgt caagcggcta 3121 gcatggctaa actcacttct gctgtaccag tactcactgc tagagatgta gctggagctg 3181 tagagttctg gactgataga ctcggattct ctagagattt cgtagaggat gatttcgctg 3241 gagtagtaag agatgatgta actctcttca tatctgctgt acaagatcaa gtagtaccag 3301 ataacactct cgcttgggta tgggtaagag gactcgatga gctctacgct gagtggtctg 3361 aggtagtatc tactaacttc agagatgctt ctggaccagc tatgactgag ataggagagc 3421 aaccgtgggg aagagagttc gctctcagag atccagctgg aaactgcgta cacttcgtag 3481 ctgaggagca agatccatgg taactcgagt gcccatttag ttggcttttc ccttgtctcg 3541 tgtcttttcc gtggaaaggt tcccggagta atctgatggc acagcaggga ggtgcgcctg 3601 caggttggtt aggaaggggg gatgatgtaa aagaagaaaa tggggggatt cgagccccgc 3661 acaggtctct caaattggat gcatagcttg agtattctat agtgtcacct aaatagcttg 3721 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac 3781 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc 3841 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg 3901 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct 3961 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac 4021 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga 4081 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat 4141 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac 4201 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct 4261 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg 4321 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg 4381 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt 4441 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg 4501 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac 4561 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga 4621 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt 4681 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt 4741 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga 4801 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc 4861 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct 4921 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata 4981 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca 5041 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga 5101 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga 5161 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg 5221 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga 5281 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt 5341 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct 5401 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca 5461 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat 5521 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga 5581 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc 5641 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg 5701 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc 5761 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt 5821 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca 5881 cctgatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggaaatt 5941 gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt 6001 aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg 6061 ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc 6121 aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca 6181 agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga 6241 tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa 6301 ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc 6361 gccgcgctta atgcgccgct acagggcgcg tccattcgcc attcaggctg cgcaactgtt 6421 gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa gggggatgtg 6481 ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgt tgtaaaacga 6541 cggccagtga attgtaatac gactcactat a // LOCUS pPLOT-nanoLuc-Puro-Puro 7009 bp ds-DNA circular SYN 04-NOV-2016 DEFINITION pPLOT-nanoLuc-Puro-Puro ACCESSION . VERSION . KEYWORDS pPLOT-nanoLuc-Puro-Puro SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7009) AUTHORS . TITLE Direct Submission JOURNAL Exported Friday, Nov 4, 2016 from SnapGene Viewer 3.2.1 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7009 /organism="synthetic DNA construct" /mol_type="other DNA" misc_feature 16..35 /note="#161" misc_feature 36..164 /note="Actin 5'" CDS 171..770 /codon_start=1 /note="Puro" /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA" misc_feature 777..925 /note="Ald3'UTR" misc_feature 932..1488 /note="PGKB 5'" CDS 1492..1581 /codon_start=1 /product="3 tandem Myc epitope tags" /note="3xMyc" /translation="EQKLISEEDLEQKLISEEDLEQKLISEEDL" misc_feature 1582..1611 /note="GS linker" misc_feature 1618..2130 /note="nanoLuc" misc_feature 2137..2166 /note="GS linker" CDS 2167..2256 /codon_start=1 /product="3 tandem Myc epitope tags" /note="3xMyc" /translation="EQKLISEEDLEQKLISEEDLEQKLISEEDL" misc_feature 2260..3335 /note="Cf PGKA/B" CDS 3342..3941 /codon_start=1 /note="Puro" /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA" misc_feature 3948..4096 /note="Ald3'UTR" promoter complement(4134..4152) /note="SP6 promoter" /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind complement(4170..4186) /note="M13 rev" /note="common sequencing primer, one of multiple similar variants" protein_bind 4194..4210 /bound_moiety="lac repressor encoded by lacI" /note="lac operator" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." promoter complement(4218..4248) /note="lac promoter" /note="promoter for the E. coli lac operon" protein_bind 4263..4284 /bound_moiety="E. coli catabolite activator protein" /note="CAP binding site" /note="CAP binding activates transcription in the presence of cAMP." rep_origin complement(4572..5160) /direction=LEFT /note="ori" /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(5331..6191) /codon_start=1 /gene="bla" /product="beta-lactamase" /note="AmpR" /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" promoter complement(6192..6296) /gene="bla" /note="AmpR promoter" rep_origin complement(6374..6829) /direction=LEFT /note="f1 ori" /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind 6970..6986 /note="M13 fwd" /note="common sequencing primer, one of multiple similar variants" promoter 6993..2 /note="T7 promoter" /note="promoter for bacteriophage T7 RNA polymerase" ORIGIN 1 gggcgaattg ggcccgtata atgcagacct gctgcaagct aggggcacag caaggtcttc 61 tgaaattcat gttttttttt tttttactct gcattgcagt ctccgctctt atttagtttt 121 gctttacgta aggtctcgtt gctgccataa aataagctct agtggaattc atgactgaat 181 acaagccaac ggttcgtctt gctacgcgag acgatgtgcc gcgggccgta cggacgctgg 241 ctgctgcgtt cgctgattat cccgccaccc gacatacggt tgacccagat cgacacattg 301 agcgagtcac agaacttcaa gagttattct taacacgggt gggcttggac attggtaagg 361 tttgggttgc ggacgatggc gcggctgttg cggtgtggac gacgccagag tcagtcgaag 421 cgggcgcagt gtttgctgag atcggtcccc gaatggcgga gcttagcggg tcacgcctcg 481 ctgcacaaca gcagatggaa ggtctgttgg caccacatag acctaaggag ccggcttggt 541 tcttagcgac agttggcgtc agtccggacc accaaggtaa ggggctgggt tcagcggttg 601 ttcttccagg agtagaggct gcggaaaggg caggtgtgcc ggcgttcctt gaaacgtctg 661 caccacgtaa ccttcctttt tacgagagat tggggtttac agtaacagca gatgttgaag 721 tgcctgaagg gccgaggaca tggtgcatga cacgtaagcc gggagcctaa ccatggtgcc 781 catttagttg gcttttccct tgtctcgtgt cttttccgtg gaaaggttcc cggagtaatc 841 tgatggcaca gcagggaggt gcgcctgcag gttggttagg aaggggggat gatgtaaaag 901 aagaaaatgg ggggattcga gccccacgcg ttcgagaccg acaagaccag aatagcgctt 961 atgtgtgtgt gtttgtgtgt gggtgtgtgt gcccatgtgt gacaaagagg ccagtgcttt 1021 ctctaaagca agagcgcgag gttgctggct aaggactcta agtccctcac agtgatggcg 1081 tgttgcattt tctaaaccgt caatccaaca cgtcggtagc atatcggttt tgtaggctct 1141 tctggatacc ttccccagta agaccgccac tgcttcgtga ctgaagcact gtaaatatca 1201 ctgaaaccgt cgcgttgctt tgtctaatta tcaccgcctt tgccagctat cgcctcaggc 1261 tgcgctcttt gactaatcca ctcaccactt ctctgccttc ttcctccggt tgttgttgtg 1321 tcctgcgtgt acatggcgcg tgtccttttc gagcaaacag ctgtcttttc tttcacgata 1381 acacactcat attaaacgcg agtattactc atcagtcaac gtcacattcc gctctgtcca 1441 cttcgacctt acacctctac ttgtccaact atcttccact tgtcaagcat ggaacagaag 1501 ctgatcagcg aagaagacct ggagcaaaag ctcattagcg aggaggacct cgagcagaag 1561 ctgattagcg aggaggacct gggttctggt agtggttccg gttccggttc taagcttatg 1621 gtatttactt tggaggattt tgtaggggat tggcgccaga cggccggcta caatctggat 1681 caggtactgg agcaaggcgg tgtttcgtcg ttgtttcaga acctgggggt gtcagtaacc 1741 cctattcaga ggatagtcct ttccggtgag aatggcctta aaattgatat ccacgtcata 1801 attccttatg aagggttgag cggggaccaa atggggcaga tagagaagat tttcaaggta 1861 gtttacccag tcgatgacca tcacttcaaa gttatattgc attatgggac tctggtcatc 1921 gacggtgtca cacctaatat gattgattac ttcggccgcc catacgaagg aatagctgtg 1981 tttgatggga agaagataac tgtgacggga actctgtgga atggtaacaa aataatcgac 2041 gagaggctta taaatcccga tggaagcctc ctgttccgtg taacgatcaa cggcgtaact 2101 ggttggcgac tgtgtgagcg gatacttgca ggatccggat caggatctgg atcaggatcg 2161 ggtagtgaac agaagctgat cagcgaagaa gacctggagc aaaagctcat tagcgaggag 2221 gacctcgagc agaagctgat tagcgaggag gacctgtaac caccaccacc actgagaatt 2281 ctgtattacg ccgttttaag agctccttcg acgccttttt gtccttcagc tgctccacgg 2341 tgactcgctt ctctctcttt ccacagtgtc tcttttttct tttcactctc tatacaaatg 2401 tgagcgacct ccttttctgt acaacggcct tccggcgtgt gcttttctca tcgacctctc 2461 ctcgcttctt gggcactcct tcatcgagca aacaagacga gggaggtgac gcgattggtg 2521 agaccactac gagtatggac ggagctgtct gataccccct gttttttatc ttgtaccccg 2581 ctctgcagtg acggtagccc gctgctgtgc ttcggtatcg ccgcttcata cgcttctctc 2641 ttttttcaac gtgcggcgct gattcaaagg ttacctcaac gcgacccgcc gctgcatcct 2701 ttgttgctcc tcttgtcgca aacaaacaaa aaacgtgctg tgctttttcc tttatcgtgt 2761 ctttttgcaa agtctagagt ttaccgacaa gaccagaata gcgcttatgt gtgtgtgttt 2821 gtgtgtgggt gtgtgtgccc atgtgtgaca aagaggccag tgctttctct aaagcaagag 2881 cgcgaggttg ctggctaagg actctaagtc cctcacagtg atggcgtgtt gcattttcta 2941 aaccgtcaat ccaacacgtc ggtagcatat cggttttgta ggctcttctg gataccttcc 3001 ccagtaagac cgccactgct tcgtgactga agcactgtaa atatcactga aaccgtcgcg 3061 ttgctttgtc taattatcac cgcctttgcc agctatcgcc tcaggctgcg ctctttgact 3121 aatccactca ccacttctct gccttcttcc tccggttgtt gttgtgtcct gcgtgtacat 3181 ggcgcgtgtc cttttcgagc aaacagctgt cttttctttc acgataacac actcatatta 3241 aacgcgagta ttactcatca gtcaacgtca cattccgctc tgtccacttc gaccttacac 3301 ctctacttgt ccaactatct tccacttgtc aagcggctag catgactgaa tacaagccaa 3361 cggttcgtct tgctacgcga gacgatgtgc cgcgggccgt acggacgctg gctgctgcgt 3421 tcgctgatta tcccgccacc cgacatacgg ttgacccaga tcgacacatt gagcgagtca 3481 cagaacttca agagttattc ttaacacggg tgggcttgga cattggtaag gtttgggttg 3541 cggacgatgg cgcggctgtt gcggtgtgga cgacgccaga gtcagtcgaa gcgggcgcag 3601 tgtttgctga gatcggtccc cgaatggcgg agcttagcgg gtcacgcctc gctgcacaac 3661 agcagatgga aggtctgttg gcaccacata gacctaagga gccggcttgg ttcttagcga 3721 cagttggcgt cagtccggac caccaaggta aggggctggg ttcagcggtt gttcttccag 3781 gagtagaggc tgcggaaagg gcaggtgtgc cggcgttcct tgaaacgtct gcaccacgta 3841 accttccttt ttacgagaga ttggggttta cagtaacagc agatgttgaa gtgcctgaag 3901 ggccgaggac atggtgcatg acacgtaagc cgggagccta actcgagtgc ccatttagtt 3961 ggcttttccc ttgtctcgtg tcttttccgt ggaaaggttc ccggagtaat ctgatggcac 4021 agcagggagg tgcgcctgca ggttggttag gaagggggga tgatgtaaaa gaagaaaatg 4081 gggggattcg agccccgcac aggtctctca aattggatgc atagcttgag tattctatag 4141 tgtcacctaa atagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat 4201 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc 4261 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga 4321 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt 4381 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg 4441 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac 4501 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg 4561 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca 4621 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc 4681 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc 4741 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag 4801 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc 4861 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca 4921 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg 4981 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg 5041 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct 5101 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa 5161 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa 5221 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa 5281 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc 5341 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga 5401 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca 5461 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc 5521 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat 5581 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc 5641 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt 5701 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc 5761 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg 5821 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt 5881 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg 5941 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga 6001 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg 6061 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg 6121 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt 6181 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc 6241 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca 6301 tttccccgaa aagtgccacc tgatgcggtg tgaaataccg cacagatgcg taaggagaaa 6361 ataccgcatc aggaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt 6421 taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa 6481 gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag 6541 aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt 6601 gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac 6661 cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag 6721 gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg 6781 cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc cattcgccat 6841 tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc 6901 tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt 6961 cacgacgttg taaaacgacg gccagtgaat tgtaatacga ctcactata // LOCUS pRM005 10919 bp ds-DNA circular SYN 22-JUN-2016 DEFINITION pRM005 ACCESSION . VERSION . KEYWORDS pRM005 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 10919) AUTHORS . TITLE Direct Submission JOURNAL Exported Friday, Oct 21, 2016 from SnapGene Viewer 3.2.1 http://www.snapgene.com COMMENT LOCUS pX330a-\Zhang\chimeric\gRNA\Cas9 8506 bp DNA circular ACCESSION pX330a-\Zhang\chimeric\gRNA\Cas9 VERSION pX330a-\Zhang\chimeric\gRNA\Cas9.0 KEYWORDS . COMMENT DISPLAY_SETUP_START| xmlns:xsi=""http://www.w3.org/2001/XMLSchema-instance"" xsi:type=""defaultDNAMotifFinderSetup""> SIMILARITY_THRESHOLD AVERAGE 75.0 NAME false 3 DIRECT COMPLEMENTARY type=""Graphic Display"" xmlns:xsi=""http://www.w3.org/2001/XMLSchema-instance"" xsi:type=""graphicSetup""> target=""3xFLAG:1251 - 1319"" text=""@N"" type=""21"" xsi:type=""label""> target=""3xFLAG:1251 - 1319"" type=""21"" xsi:type=""line""> target=""3xFLAG:1251 - 1319"" type=""21"" xsi:type=""symbol""> target=""R-ITR:5769 - 5909"" text=""@N"" type=""21"" xsi:type=""label""> target=""R-ITR:5769 - 5909"" type=""21"" xsi:type=""line""> target=""R-ITR:5769 - 5909"" type=""21"" xsi:type=""symbol""> text=""@N"" type=""21"" xsi:type=""label""> type=""21"" xsi:type=""line""> target=""NLS:1320 - 1370"" type=""21"" xsi:type=""symbol""> text=""@N"" type=""21"" xsi:type=""label""> type=""21"" xsi:type=""line""> target=""NLS:5472 - 5519"" type=""21"" xsi:type=""symbol""> target=""Chimeric guide RNA scaffold:268 - 343"" text=""@N"" type=""21"" xsi:type=""label""> target=""Chimeric guide RNA scaffold:268 - 343"" type=""21"" xsi:type=""line""> shape=""1"" target=""Chimeric guide RNA scaffold:268 - 343"" type=""21"" xsi:type=""symbol""> target=""AmpR:6826 - 7683"" text=""@N"" type=""21"" xsi:type=""label""> target=""AmpR:6826 - 7683"" type=""21"" xsi:type=""line""> target=""AmpR:6826 - 7683"" type=""21"" xsi:type=""symbol""> target=""TSS (+1):250 - 250"" text=""@N"" type=""21"" xsi:type=""label""> target=""TSS (+1):250 - 250"" type=""21"" xsi:type=""line""> target=""TSS (+1):250 - 250"" type=""21"" xsi:type=""symbol""> target=""Source_1:1 - 8506"" text=""@N"" type=""98"" xsi:type=""label""> target=""Source_1:1 - 8506"" type=""98"" xsi:type=""line""> target=""Source_1:1 - 8506"" type=""98"" xsi:type=""symbol""> target=""AvaI (3634):3633 - 3638"" text=""@N (@S)"" type=""68"" xsi:type=""label""> target=""AvaI (3634):3633 - 3638"" type=""68"" xsi:type=""line""> target=""AvaI (3634):3633 - 3638"" type=""68"" xsi:type=""symbol""> target=""PstI (2162):2157 - 2162"" text=""@N (@S)"" type=""68"" xsi:type=""label""> target=""PstI (2162):2157 - 2162"" type=""68"" xsi:type=""line""> target=""PstI (2162):2157 - 2162"" type=""68"" xsi:type=""symbol""> target=""XmaI (5855):5854 - 5859"" text=""@N (@S)"" type=""68"" xsi:type=""label""> target=""XmaI (5855):5854 - 5859"" type=""68"" xsi:type=""line""> target=""XmaI (5855):5854 - 5859"" type=""68"" xsi:type=""symbol""> target=""AvaI (6322):6321 - 6326"" text=""@N (@S)"" type=""68"" xsi:type=""label""> target=""AvaI (6322):6321 - 6326"" type=""68"" xsi:type=""line""> target=""AvaI (6322):6321 - 6326"" type=""68"" xsi:type=""symbol""> target=""NcoI (1250):1249 - 1254"" text=""@N (@S)"" type=""68"" xsi:type=""label""> target=""NcoI (1250):1249 - 1254"" type=""68"" xsi:type=""line""> target=""NcoI (1250):1249 - 1254"" type=""68"" xsi:type=""symbol""> target=""NcoI (722):721 - 726"" text=""@N (@S)"" type=""68"" xsi:type=""label""> target=""NcoI (722):721 - 726"" type=""68"" xsi:type=""line""> target=""NcoI (722):721 - 726"" type=""68"" xsi:type=""symbol""> target=""PstI (418):413 - 418"" text=""@N (@S)"" type=""68"" xsi:type=""label""> target=""PstI (418):413 - 418"" type=""68"" xsi:type=""line""> target=""PstI (418):413 - 418"" type=""68"" xsi:type=""symbol""> target=""PstI (3818):3813 - 3818"" text=""@N (@S)"" type=""68"" xsi:type=""label""> target=""PstI (3818):3813 - 3818"" type=""68"" xsi:type=""line""> target=""PstI (3818):3813 - 3818"" type=""68"" xsi:type=""symbol""> target=""AvaI (5855):5854 - 5859"" text=""@N (@S)"" type=""68"" xsi:type=""label""> target=""AvaI (5855):5854 - 5859"" type=""68"" xsi:type=""line""> target=""AvaI (5855):5854 - 5859"" type=""68"" xsi:type=""symbol""> target=""XmaI (5866):5865 - 5870"" text=""@N (@S)"" type=""68"" xsi:type=""label""> target=""XmaI (5866):5865 - 5870"" type=""68"" xsi:type=""line""> target=""XmaI (5866):5865 - 5870"" type=""68"" xsi:type=""symbol""> target=""SmaI (5868):5865 - 5870"" text=""@N (@S)"" type=""68"" xsi:type=""label""> target=""SmaI (5868):5865 - 5870"" type=""68"" xsi:type=""line""> FEATURES Location/Qualifiers source 1..10919 /organism="synthetic DNA construct" /mol_type="other DNA" misc_feature 148..815 /note="rep_origin" /note=" rep_origin 7834..8501 /label=pUC ori /label=pUC ori /vntifkey=""33"" /dnas_title=""pUC ori"" /original_location=""7834..8501"" " misc_feature 841..1184 /note="5' target" misc_feature 1185..1741 /note="PGKB 5'" misc_feature 1760..1828 /note=" misc_feature 1251..1319 /label=3xFLAG /label=3xFLAG /vntifkey=""21"" /note=""3xFLAG"" /original_location=""1251..1319"" " CDS 1829..6031 /codon_start=1 /note="Cas9 (replace with ORF of Addback)" /translation="MAPKKKRKVGIHGVPAADKKYSIGLDIGTNSVGWAVITDEYKVPS KKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSN EMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTD KADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGV DAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQ LSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKR YDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKM DGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKN LPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVT VKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDI VLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKT ILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGI LQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQIL KEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNK VLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKA GFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFY KVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKA TAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQ VNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVE KGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENG RKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDE IIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYF DTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDKRPAATKKAGQAKKKK" CDS 1835..1858 /codon_start=1 /note="SV40 NLS" /translation="PKKKRKVG" CDS 1841..1855 /codon_start=1 /note="NLS" /translation="KKRKV" misc_feature 6039..6562 /note="CfPGKA-3'" misc_feature 6563..7124 /note="CfPGKB-5'" CDS 7130..7501 /codon_start=1 /note="BLE" /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR EFALRDPAGNCVHFVAEEQD" misc_feature 7534..8763 /note="CfGSPS 3'UTR" misc_feature 8778..9111 /note="3' target" misc_feature 9234..9540 /note="rep_origin" /note=" rep_origin 6001..6307 /label=f1 Origin /label=f1 Origin /vntifkey=""33"" /dnas_title=""f1 Origin"" /original_location=""6001..6307"" " misc_feature 9808..9830 /note="pGEX-3 Primer" misc_feature 9989..10017 /note="AmpR Promoter" CDS 10059..10919 /codon_start=1 /note="AmpR" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" ORIGIN 1 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt 61 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag 121 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct 181 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt 241 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg 301 cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct 361 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc 421 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg 481 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa 541 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg 601 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg 661 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga 721 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt 781 ttacggttcc tggccttttg ctggcctttt gctcacatgt aagcttttaa ttaaggtacc 841 caaagaaacg gcgcttcctc gtccgcatgt gtttgcggga catgacgccg cgccactgcc 901 acgccttttt cttgcctgct gtgaggagcg gccgcggccc ttggcgctcc cccccccgcc 961 tgcgtctccg ccagcggcgg ccacctgtgt cacaccccat ctcccccgct ctctctcccc 1021 cgtctctcct ggtctcgaaa caacccgaca ttcggaaata tgtttgcacg gcgttgcgcg 1081 cgtgtcacac acgcacacac acaccgacca gtaaccgtcc cccaacaccc cctcctccgc 1141 acaaagcata ccctttcgtt tcgcctttcg ccactctgcc atcctcgaga ccgacaagac 1201 cagaatagcg cttatgtgtg tgtgtttgtg tgtgggtgtg tgtgcccatg tgtgacaaag 1261 aggccagtgc tttctctaaa gcaagagcgc gaggttgctg gctaaggact ctaagtccct 1321 cacagtgatg gcgtgttgca ttttctaaac cgtcaatcca acacgtcggt agcatatcgg 1381 ttttgtaggc tcttctggat accttcccca gtaagaccgc cactgcttcg tgactgaagc 1441 actgtaaata tcactgaaac cgtcgcgttg ctttgtctaa ttatcaccgc ctttgccagc 1501 tatcgcctca ggctgcgctc tttgactaat ccactcacca cttctctgcc ttcttcctcc 1561 ggttgttgtt gtgtcctgcg tgtacatggc gcgtgtcctt ttcgagcaaa cagctgtctt 1621 ttctttcacg ataacacact catattaaac gcgagtatta ctcatcagtc aacgtcacat 1681 tccgctctgt ccacttcgac cttacacctc tacttgtcca actatcttcc acttgtcaag 1741 cactagtacc ggtgccacca tggactataa ggaccacgac ggagactaca aggatcatga 1801 tattgattac aaagacgatg acgataagat ggccccaaag aagaagcgga aggtcggtat 1861 ccacggagtc ccagcagccg acaagaagta cagcatcggc ctggacatcg gcaccaactc 1921 tgtgggctgg gccgtgatca ccgacgagta caaggtgccc agcaagaaat tcaaggtgct 1981 gggcaacacc gaccggcaca gcatcaagaa gaacctgatc ggagccctgc tgttcgacag 2041 cggcgaaaca gccgaggcca cccggctgaa gagaaccgcc agaagaagat acaccagacg 2101 gaagaaccgg atctgctatc tgcaagagat cttcagcaac gagatggcca aggtggacga 2161 cagcttcttc cacagactgg aagagtcctt cctggtggaa gaggataaga agcacgagcg 2221 gcaccccatc ttcggcaaca tcgtggacga ggtggcctac cacgagaagt accccaccat 2281 ctaccacctg agaaagaaac tggtggacag caccgacaag gccgacctgc ggctgatcta 2341 tctggccctg gcccacatga tcaagttccg gggccacttc ctgatcgagg gcgacctgaa 2401 ccccgacaac agcgacgtgg acaagctgtt catccagctg gtgcagacct acaaccagct 2461 gttcgaggaa aaccccatca acgccagcgg cgtggacgcc aaggccatcc tgtctgccag 2521 actgagcaag agcagacggc tggaaaatct gatcgcccag ctgcccggcg agaagaagaa 2581 tggcctgttc ggaaacctga ttgccctgag cctgggcctg acccccaact tcaagagcaa 2641 cttcgacctg gccgaggatg ccaaactgca gctgagcaag gacacctacg acgacgacct 2701 ggacaacctg ctggcccaga tcggcgacca gtacgccgac ctgtttctgg ccgccaagaa 2761 cctgtccgac gccatcctgc tgagcgacat cctgagagtg aacaccgaga tcaccaaggc 2821 ccccctgagc gcctctatga tcaagagata cgacgagcac caccaggacc tgaccctgct 2881 gaaagctctc gtgcggcagc agctgcctga gaagtacaaa gagattttct tcgaccagag 2941 caagaacggc tacgccggct acattgacgg cggagccagc caggaagagt tctacaagtt 3001 catcaagccc atcctggaaa agatggacgg caccgaggaa ctgctcgtga agctgaacag 3061 agaggacctg ctgcggaagc agcggacctt cgacaacggc agcatccccc accagatcca 3121 cctgggagag ctgcacgcca ttctgcggcg gcaggaagat ttttacccat tcctgaagga 3181 caaccgggaa aagatcgaga agatcctgac cttccgcatc ccctactacg tgggccctct 3241 ggccagggga aacagcagat tcgcctggat gaccagaaag agcgaggaaa ccatcacccc 3301 ctggaacttc gaggaagtgg tggacaaggg cgcttccgcc cagagcttca tcgagcggat 3361 gaccaacttc gataagaacc tgcccaacga gaaggtgctg cccaagcaca gcctgctgta 3421 cgagtacttc accgtgtata acgagctgac caaagtgaaa tacgtgaccg agggaatgag 3481 aaagcccgcc ttcctgagcg gcgagcagaa aaaggccatc gtggacctgc tgttcaagac 3541 caaccggaaa gtgaccgtga agcagctgaa agaggactac ttcaagaaaa tcgagtgctt 3601 cgactccgtg gaaatctccg gcgtggaaga tcggttcaac gcctccctgg gcacatacca 3661 cgatctgctg aaaattatca aggacaagga cttcctggac aatgaggaaa acgaggacat 3721 tctggaagat atcgtgctga ccctgacact gtttgaggac agagagatga tcgaggaacg 3781 gctgaaaacc tatgcccacc tgttcgacga caaagtgatg aagcagctga agcggcggag 3841 atacaccggc tggggcaggc tgagccggaa gctgatcaac ggcatccggg acaagcagtc 3901 cggcaagaca atcctggatt tcctgaagtc cgacggcttc gccaacagaa acttcatgca 3961 gctgatccac gacgacagcc tgacctttaa agaggacatc cagaaagccc aggtgtccgg 4021 ccagggcgat agcctgcacg agcacattgc caatctggcc ggcagccccg ccattaagaa 4081 gggcatcctg cagacagtga aggtggtgga cgagctcgtg aaagtgatgg gccggcacaa 4141 gcccgagaac atcgtgatcg aaatggccag agagaaccag accacccaga agggacagaa 4201 gaacagccgc gagagaatga agcggatcga agagggcatc aaagagctgg gcagccagat 4261 cctgaaagaa caccccgtgg aaaacaccca gctgcagaac gagaagctgt acctgtacta 4321 cctgcagaat gggcgggata tgtacgtgga ccaggaactg gacatcaacc ggctgtccga 4381 ctacgatgtg gaccatatcg tgcctcagag ctttctgaag gacgactcca tcgacaacaa 4441 ggtgctgacc agaagcgaca agaaccgggg caagagcgac aacgtgccct ccgaagaggt 4501 cgtgaagaag atgaagaact actggcggca gctgctgaac gccaagctga ttacccagag 4561 aaagttcgac aatctgacca aggccgagag aggcggcctg agcgaactgg ataaggccgg 4621 cttcatcaag agacagctgg tggaaacccg gcagatcaca aagcacgtgg cacagatcct 4681 ggactcccgg atgaacacta agtacgacga gaatgacaag ctgatccggg aagtgaaagt 4741 gatcaccctg aagtccaagc tggtgtccga tttccggaag gatttccagt tttacaaagt 4801 gcgcgagatc aacaactacc accacgccca cgacgcctac ctgaacgccg tcgtgggaac 4861 cgccctgatc aaaaagtacc ctaagctgga aagcgagttc gtgtacggcg actacaaggt 4921 gtacgacgtg cggaagatga tcgccaagag cgagcaggaa atcggcaagg ctaccgccaa 4981 gtacttcttc tacagcaaca tcatgaactt tttcaagacc gagattaccc tggccaacgg 5041 cgagatccgg aagcggcctc tgatcgagac aaacggcgaa accggggaga tcgtgtggga 5101 taagggccgg gattttgcca ccgtgcggaa agtgctgagc atgccccaag tgaatatcgt 5161 gaaaaagacc gaggtgcaga caggcggctt cagcaaagag tctatcctgc ccaagaggaa 5221 cagcgataag ctgatcgcca gaaagaagga ctgggaccct aagaagtacg gcggcttcga 5281 cagccccacc gtggcctatt ctgtgctggt ggtggccaaa gtggaaaagg gcaagtccaa 5341 gaaactgaag agtgtgaaag agctgctggg gatcaccatc atggaaagaa gcagcttcga 5401 gaagaatccc atcgactttc tggaagccaa gggctacaaa gaagtgaaaa aggacctgat 5461 catcaagctg cctaagtact ccctgttcga gctggaaaac ggccggaaga gaatgctggc 5521 ctctgccggc gaactgcaga agggaaacga actggccctg ccctccaaat atgtgaactt 5581 cctgtacctg gccagccact atgagaagct gaagggctcc cccgaggata atgagcagaa 5641 acagctgttt gtggaacagc acaagcacta cctggacgag atcatcgagc agatcagcga 5701 gttctccaag agagtgatcc tggccgacgc taatctggac aaagtgctgt ccgcctacaa 5761 caagcaccgg gataagccca tcagagagca ggccgagaat atcatccacc tgtttaccct 5821 gaccaatctg ggagcccctg ccgccttcaa gtactttgac accaccatcg accggaagag 5881 gtacaccagc accaaagagg tgctggacgc caccctgatc caccagagca tcaccggcct 5941 gtacgagaca cggatcgacc tgtctcagct gggaggcgac aaaaggccgg cggccacgaa 6001 aaaggccggc caggcaaaaa agaaaaagta agaattcctc gagcaccacc accaccacca 6061 ctgagaattc tgtattacgc cgttttaaga gctccttcga cgcctttttg tccttcagct 6121 gctccacggt gactcgcttc tctctctttc cacagtgtct cttttttctt ttcactctct 6181 atacaaatgt gagcgacctc cttttctgta caacggcctt ccggcgtgtg cttttctcat 6241 cgacctctcc tcgcttcttg ggcactcctt catcgagcaa acaagacgag ggaggtgacg 6301 cgattggtga gaccactacg agtatggacg gagctgtctg ataccccctg ttttttatct 6361 tgtaccccgc tctgcagtga cggtagcccg ctgctgtgct tcggtatcgc cgcttcatac 6421 gcttctctct tttttcaacg tgcggcgctg attcaaaggt tacctcaacg cgacccgccg 6481 ctgcatcctt tgttgctcct cttgtcgcaa acaaacaaaa aacgtgctgt gctttttcct 6541 ttatcgtgtc tttttgcaaa gtctagagtt taccgacaag accagaatag cgcttatgtg 6601 tgtgtgtttg tgtgtgggtg tgtgtgccca tgtgtgacaa agaggccagt gctttctcta 6661 aagcaagagc gcgaggttgc tggctaagga ctctaagtcc ctcacagtga tggcgtgttg 6721 cattttctaa accgtcaatc caacacgtcg gtagcatatc ggttttgtag gctcttctgg 6781 ataccttccc cagtaagacc gccactgctt cgtgactgaa gcactgtaaa tatcactgaa 6841 accgtcgcgt tgctttgtct aattatcacc gcctttgcca gctatcgcct caggctgcgc 6901 tctttgacta atccactcac cacttctctg ccttcttcct ccggttgttg ttgtgtcctg 6961 cgtgtacatg gcgcgtgtcc ttttcgagca aacagctgtc ttttctttca cgataacaca 7021 ctcatattaa acgcgagtat tactcatcag tcaacgtcac attccgctct gtccacttcg 7081 accttacacc tctacttgtc caactatctt ccacttgtca agcgtcgaca tggccaagtt 7141 gaccagtgcc gttccggtgc tcaccgcgcg cgacgtcgcc ggagcggtcg agttctggac 7201 cgaccggctc gggttctccc gggacttcgt ggaggacgac ttcgccggtg tggtccggga 7261 cgacgtgacc ctgttcatca gcgcggtcca ggaccaggtg gtgccggaca acaccctggc 7321 ctgggtgtgg gtgcgcggcc tggacgagct gtacgccgag tggtcggagg tcgtgtccac 7381 gaacttccgg gacgcctccg ggccggccat gaccgagatc ggcgagcagc cgtgggggcg 7441 ggagttcgcc ctgcgcgacc cggccggcaa ctgcgtgcac ttcgtggccg aggagcagga 7501 ctgaggagga tcccgtatct cgaggatcta gaggatccag caggcggaga aagagggaag 7561 tataaggcgg acgcataggt cggagtatcg agaaaaagag gcagagatgg gtggtggcgg 7621 agcgcccctc tctgcccttc tctgttttac tgttcccgcc acgtcgccaa ctcctctttt 7681 tgtttgctct aatgtcggtg ctatcctctt cctcttttta ctctccgttt ttcttccttc 7741 cgttttgtct tgttatcacc gttatttttc ttcttctttc ttagccgatt tgggtctcct 7801 gcctacggca gcggtgatga gtcgcacttc cgtccccctt tcctctccgt aagtactccc 7861 tcgatgcctc aggcgcttct attttgcggc actgtgctga ccacctccca cgtgtgcagt 7921 gagagcgcca gagacattca ggagaagagg gaagagggga agtaaatacc aaagcgagga 7981 agatgtcttt ctcgctgctt ctcatcctgt tgaccggtgt gcacggcggt gtgtcctctc 8041 ggcttccctc ctccttccct cgctcccctt tttctgtgtt ttttctttct aacatgattg 8101 cgcctgctct ttttgccccg cagcgtcgca gtgggtgtca cctgcgaccg ccgtacgttt 8161 tactatatac atatttgttt ctgtgctgat gccatttgtc cgctactcca tatcgtgcgt 8221 gtttgttttc cagtggtgac gccttccgca tcattttaat caattcgagg cgaaaagggt 8281 gatttgagtg ttggagtggg cttttttatt tatttgactc caaacatctt ttcttttccc 8341 ggtatcttcg ggtccgtcac gcaggtcggt gggtgtgtcc gagtgcgctt ctttactcac 8401 gtgccgctgc gcacatacac agattttaaa agcacgcata cacgatctgt gccttcaaaa 8461 ctaatcaaca aaaacaaaaa tacaaaaaag acaagaataa gaggtcagca acgcacccac 8521 ggctcctttc ttcctgatcc acgtcgtggc cgctgtacgc tctcaaacac gctttggcgc 8581 tgatgcgtgc ctttatacaa agaacaaaga agaagcgaaa aacgggagcg tgttgctttg 8641 gggatgtatg cgtgccacag ttctgcggta caattcgtaa tcatggtcat agctgtttcc 8701 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg 8761 taaatgcatg cggccgcaca gtgtgtgggt gaggtgcgcg aaggtgtgtc tgtcggtggg 8821 ggagctcgcg cggtgtgtgc gttggcggct cagcggcccc cctcgcgtct ctcacgccgg 8881 cccgcctgtc cccctccccc acataaaagg acgcagcgcg ctcagctttt gtgtgcatgt 8941 atgctatcgc tggtcgcgtg tgctgcgcct ctgtatcttc ttttgaatct ttcccttttt 9001 ttcgctttct tttatcttcc tcccgtgtac cgcagttgat ggatcaggcg ccctgttgca 9061 acttgggccg ccgacggtgc tggtgcgagg cagcaggcca gcatgttgcc gagatcttta 9121 attaaaatcc ctagcctgca ggggcgcctg atgcggtatt ttctccttac gcatctgtgc 9181 ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa 9241 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc 9301 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag 9361 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca 9421 aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc 9481 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa 9541 cactcaaccc tatctcgggc tattcttttg atttataagg gattttgccg atttcggcct 9601 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa 9661 cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc 9721 agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat 9781 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt 9841 catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg 9901 tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa 9961 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac 10021 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg 10081 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc 10141 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg 10201 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga 10261 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc 10321 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag 10381 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga 10441 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg 10501 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga 10561 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt 10621 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact 10681 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt 10741 ttattgctga taaatctgga gccggtgagc gtggaagccg cggtatcatt gcagcactgg 10801 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta 10861 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaa // LOCUS pRM006 11572 bp ds-DNA circular SYN 19-SEP-2016 DEFINITION pRM006 ACCESSION . VERSION . KEYWORDS pRM006.gb SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 11572) AUTHORS . TITLE Direct Submission JOURNAL Exported Monday, Sep 19, 2016 from SnapGene Viewer 3.2.1 http://www.snapgene.com COMMENT LOCUS pX330a-\Zhang\chimeric\gRNA\Cas9 8506 bp DNA circular ACCESSION pX330a-\Zhang\chimeric\gRNA\Cas9 VERSION pX330a-\Zhang\chimeric\gRNA\Cas9.0 KEYWORDS . COMMENT DISPLAY_SETUP_START| xmlns:xsi=""http://www.w3.org/2001/XMLSchema-instance"" xsi:type=""defaultDNAMotifFinderSetup""> SIMILARITY_THRESHOLD AVERAGE 75.0 NAME false 3 DIRECT COMPLEMENTARY type=""Graphic Display"" xmlns:xsi=""http://www.w3.org/2001/XMLSchema-instance"" xsi:type=""graphicSetup""> target=""3xFLAG:1251 - 1319"" text=""@N"" type=""21"" xsi:type=""label""> target=""3xFLAG:1251 - 1319"" type=""21"" xsi:type=""line""> target=""3xFLAG:1251 - 1319"" type=""21"" xsi:type=""symbol""> target=""R-ITR:5769 - 5909"" text=""@N"" type=""21"" xsi:type=""label""> target=""R-ITR:5769 - 5909"" type=""21"" xsi:type=""line""> target=""R-ITR:5769 - 5909"" type=""21"" xsi:type=""symbol""> text=""@N"" type=""21"" xsi:type=""label""> type=""21"" xsi:type=""line""> target=""NLS:1320 - 1370"" type=""21"" xsi:type=""symbol""> text=""@N"" type=""21"" xsi:type=""label""> type=""21"" xsi:type=""line""> target=""NLS:5472 - 5519"" type=""21"" xsi:type=""symbol""> target=""Chimeric guide RNA scaffold:268 - 343"" text=""@N"" type=""21"" xsi:type=""label""> target=""Chimeric guide RNA scaffold:268 - 343"" type=""21"" xsi:type=""line""> shape=""1"" target=""Chimeric guide RNA scaffold:268 - 343"" type=""21"" xsi:type=""symbol""> target=""AmpR:6826 - 7683"" text=""@N"" type=""21"" xsi:type=""label""> target=""AmpR:6826 - 7683"" type=""21"" xsi:type=""line""> target=""AmpR:6826 - 7683"" type=""21"" xsi:type=""symbol""> target=""TSS (+1):250 - 250"" text=""@N"" type=""21"" xsi:type=""label""> target=""TSS (+1):250 - 250"" type=""21"" xsi:type=""line""> target=""TSS (+1):250 - 250"" type=""21"" xsi:type=""symbol""> target=""Source_1:1 - 8506"" text=""@N"" type=""98"" xsi:type=""label""> target=""Source_1:1 - 8506"" type=""98"" xsi:type=""line""> target=""Source_1:1 - 8506"" type=""98"" xsi:type=""symbol""> target=""AvaI (3634):3633 - 3638"" text=""@N (@S)"" type=""68"" xsi:type=""label""> target=""AvaI (3634):3633 - 3638"" type=""68"" xsi:type=""line""> target=""AvaI (3634):3633 - 3638"" type=""68"" xsi:type=""symbol""> target=""PstI (2162):2157 - 2162"" text=""@N (@S)"" type=""68"" xsi:type=""label""> target=""PstI (2162):2157 - 2162"" type=""68"" xsi:type=""line""> target=""PstI (2162):2157 - 2162"" type=""68"" xsi:type=""symbol""> target=""XmaI (5855):5854 - 5859"" text=""@N (@S)"" type=""68"" xsi:type=""label""> target=""XmaI (5855):5854 - 5859"" type=""68"" xsi:type=""line""> target=""XmaI (5855):5854 - 5859"" type=""68"" xsi:type=""symbol""> target=""AvaI (6322):6321 - 6326"" text=""@N (@S)"" type=""68"" xsi:type=""label""> target=""AvaI (6322):6321 - 6326"" type=""68"" xsi:type=""line""> target=""AvaI (6322):6321 - 6326"" type=""68"" xsi:type=""symbol""> target=""NcoI (1250):1249 - 1254"" text=""@N (@S)"" type=""68"" xsi:type=""label""> target=""NcoI (1250):1249 - 1254"" type=""68"" xsi:type=""line""> target=""NcoI (1250):1249 - 1254"" type=""68"" xsi:type=""symbol""> target=""NcoI (722):721 - 726"" text=""@N (@S)"" type=""68"" xsi:type=""label""> target=""NcoI (722):721 - 726"" type=""68"" xsi:type=""line""> target=""NcoI (722):721 - 726"" type=""68"" xsi:type=""symbol""> target=""PstI (418):413 - 418"" text=""@N (@S)"" type=""68"" xsi:type=""label""> target=""PstI (418):413 - 418"" type=""68"" xsi:type=""line""> target=""PstI (418):413 - 418"" type=""68"" xsi:type=""symbol""> target=""PstI (3818):3813 - 3818"" text=""@N (@S)"" type=""68"" xsi:type=""label""> target=""PstI (3818):3813 - 3818"" type=""68"" xsi:type=""line""> target=""PstI (3818):3813 - 3818"" type=""68"" xsi:type=""symbol""> target=""AvaI (5855):5854 - 5859"" text=""@N (@S)"" type=""68"" xsi:type=""label""> target=""AvaI (5855):5854 - 5859"" type=""68"" xsi:type=""line""> target=""AvaI (5855):5854 - 5859"" type=""68"" xsi:type=""symbol""> target=""XmaI (5866):5865 - 5870"" text=""@N (@S)"" type=""68"" xsi:type=""label""> target=""XmaI (5866):5865 - 5870"" type=""68"" xsi:type=""line""> target=""XmaI (5866):5865 - 5870"" type=""68"" xsi:type=""symbol""> target=""SmaI (5868):5865 - 5870"" text=""@N (@S)"" type=""68"" xsi:type=""label""> target=""SmaI (5868):5865 - 5870"" type=""68"" xsi:type=""line""> FEATURES Location/Qualifiers source 1..11572 /organism="synthetic DNA construct" /mol_type="other DNA" misc_feature 148..815 /note="rep_origin" /note=" rep_origin 7834..8501 /label=pUC ori /label=pUC ori /vntifkey=""33"" /dnas_title=""pUC ori"" /original_location=""7834..8501"" " misc_feature 841..1184 /note="5' target - beta tubulin" /note="LmjF.33:339,096..339,438" misc_feature 1185..1741 /note="PGKB 5'" CDS 1763..1783 /codon_start=1 /note="FLAG" /translation="DYKDHDG" CDS 1784..1804 /codon_start=1 /note="FLAG" /translation="DYKDHDI" CDS 1805..1828 /codon_start=1 /note="FLAG" /translation="DYKDDDDK" CDS 1835..1855 /codon_start=1 /product="nuclear localization signal of SV40 large T antigen" /note="SV40 NLS" /translation="PKKKRKV" CDS 1880..5980 /codon_start=1 /product="Cas9 (Csn1) endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /note="Cas9" /note="generates RNA-guided double strand breaks in DNA" /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGD" CDS 5981..6028 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /note="nucleoplasmin NLS" /translation="KRPAATKKAGQAKKKK" misc_feature 6039..6562 /note="CfPGKA-3'" CDS 6044..6061 /codon_start=1 /product="6xHis affinity tag" /note="6xHis" /translation="HHHHHH" misc_feature 6563..7124 /note="CfPGKB 5'UTR" CDS 7133..8158 /codon_start=1 /note="hygR" /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK E" misc_feature 8187..9416 /note="3'UTR" primer_bind complement(9339..9355) /note="M13 rev" /note="common sequencing primer, one of multiple similar variants" protein_bind 9363..9379 /bound_moiety="lac repressor encoded by lacI" /note="lac operator" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." promoter complement(9387..9417) /note="lac promoter" /note="promoter for the E. coli lac operon" misc_feature 9431..9764 /note="3' target - beta tubulin" /note="LmjF.33:338,446..343,445 -" rep_origin 9870..10325 /direction=RIGHT /note="f1 ori" /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" misc_feature 10461..10483 /note="pGEX-3 Primer" promoter 10607..10711 /gene="bla" /note="AmpR promoter" misc_feature 10642..10670 /note="AmpR Promoter" CDS 10712..11572 /codon_start=1 /note="AmpR" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" ORIGIN 1 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt 61 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag 121 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct 181 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt 241 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg 301 cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct 361 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc 421 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg 481 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa 541 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg 601 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg 661 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga 721 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt 781 ttacggttcc tggccttttg ctggcctttt gctcacatgt aagcttttaa ttaaggtacc 841 caaagaaacg gcgcttcctc gtccgcatgt gtttgcggga catgacgccg cgccactgcc 901 acgccttttt cttgcctgct gtgaggagcg gccgcggccc ttggcgctcc cccccccgcc 961 tgcgtctccg ccagcggcgg ccacctgtgt cacaccccat ctcccccgct ctctctcccc 1021 cgtctctcct ggtctcgaaa caacccgaca ttcggaaata tgtttgcacg gcgttgcgcg 1081 cgtgtcacac acgcacacac acaccgacca gtaaccgtcc cccaacaccc cctcctccgc 1141 acaaagcata ccctttcgtt tcgcctttcg ccactctgcc atcctcgaga ccgacaagac 1201 cagaatagcg cttatgtgtg tgtgtttgtg tgtgggtgtg tgtgcccatg tgtgacaaag 1261 aggccagtgc tttctctaaa gcaagagcgc gaggttgctg gctaaggact ctaagtccct 1321 cacagtgatg gcgtgttgca ttttctaaac cgtcaatcca acacgtcggt agcatatcgg 1381 ttttgtaggc tcttctggat accttcccca gtaagaccgc cactgcttcg tgactgaagc 1441 actgtaaata tcactgaaac cgtcgcgttg ctttgtctaa ttatcaccgc ctttgccagc 1501 tatcgcctca ggctgcgctc tttgactaat ccactcacca cttctctgcc ttcttcctcc 1561 ggttgttgtt gtgtcctgcg tgtacatggc gcgtgtcctt ttcgagcaaa cagctgtctt 1621 ttctttcacg ataacacact catattaaac gcgagtatta ctcatcagtc aacgtcacat 1681 tccgctctgt ccacttcgac cttacacctc tacttgtcca actatcttcc acttgtcaag 1741 cactagtacc ggtgccacca tggactataa ggaccacgac ggagactaca aggatcatga 1801 tattgattac aaagacgatg acgataagat ggccccaaag aagaagcgga aggtcggtat 1861 ccacggagtc ccagcagccg acaagaagta cagcatcggc ctggacatcg gcaccaactc 1921 tgtgggctgg gccgtgatca ccgacgagta caaggtgccc agcaagaaat tcaaggtgct 1981 gggcaacacc gaccggcaca gcatcaagaa gaacctgatc ggagccctgc tgttcgacag 2041 cggcgaaaca gccgaggcca cccggctgaa gagaaccgcc agaagaagat acaccagacg 2101 gaagaaccgg atctgctatc tgcaagagat cttcagcaac gagatggcca aggtggacga 2161 cagcttcttc cacagactgg aagagtcctt cctggtggaa gaggataaga agcacgagcg 2221 gcaccccatc ttcggcaaca tcgtggacga ggtggcctac cacgagaagt accccaccat 2281 ctaccacctg agaaagaaac tggtggacag caccgacaag gccgacctgc ggctgatcta 2341 tctggccctg gcccacatga tcaagttccg gggccacttc ctgatcgagg gcgacctgaa 2401 ccccgacaac agcgacgtgg acaagctgtt catccagctg gtgcagacct acaaccagct 2461 gttcgaggaa aaccccatca acgccagcgg cgtggacgcc aaggccatcc tgtctgccag 2521 actgagcaag agcagacggc tggaaaatct gatcgcccag ctgcccggcg agaagaagaa 2581 tggcctgttc ggaaacctga ttgccctgag cctgggcctg acccccaact tcaagagcaa 2641 cttcgacctg gccgaggatg ccaaactgca gctgagcaag gacacctacg acgacgacct 2701 ggacaacctg ctggcccaga tcggcgacca gtacgccgac ctgtttctgg ccgccaagaa 2761 cctgtccgac gccatcctgc tgagcgacat cctgagagtg aacaccgaga tcaccaaggc 2821 ccccctgagc gcctctatga tcaagagata cgacgagcac caccaggacc tgaccctgct 2881 gaaagctctc gtgcggcagc agctgcctga gaagtacaaa gagattttct tcgaccagag 2941 caagaacggc tacgccggct acattgacgg cggagccagc caggaagagt tctacaagtt 3001 catcaagccc atcctggaaa agatggacgg caccgaggaa ctgctcgtga agctgaacag 3061 agaggacctg ctgcggaagc agcggacctt cgacaacggc agcatccccc accagatcca 3121 cctgggagag ctgcacgcca ttctgcggcg gcaggaagat ttttacccat tcctgaagga 3181 caaccgggaa aagatcgaga agatcctgac cttccgcatc ccctactacg tgggccctct 3241 ggccagggga aacagcagat tcgcctggat gaccagaaag agcgaggaaa ccatcacccc 3301 ctggaacttc gaggaagtgg tggacaaggg cgcttccgcc cagagcttca tcgagcggat 3361 gaccaacttc gataagaacc tgcccaacga gaaggtgctg cccaagcaca gcctgctgta 3421 cgagtacttc accgtgtata acgagctgac caaagtgaaa tacgtgaccg agggaatgag 3481 aaagcccgcc ttcctgagcg gcgagcagaa aaaggccatc gtggacctgc tgttcaagac 3541 caaccggaaa gtgaccgtga agcagctgaa agaggactac ttcaagaaaa tcgagtgctt 3601 cgactccgtg gaaatctccg gcgtggaaga tcggttcaac gcctccctgg gcacatacca 3661 cgatctgctg aaaattatca aggacaagga cttcctggac aatgaggaaa acgaggacat 3721 tctggaagat atcgtgctga ccctgacact gtttgaggac agagagatga tcgaggaacg 3781 gctgaaaacc tatgcccacc tgttcgacga caaagtgatg aagcagctga agcggcggag 3841 atacaccggc tggggcaggc tgagccggaa gctgatcaac ggcatccggg acaagcagtc 3901 cggcaagaca atcctggatt tcctgaagtc cgacggcttc gccaacagaa acttcatgca 3961 gctgatccac gacgacagcc tgacctttaa agaggacatc cagaaagccc aggtgtccgg 4021 ccagggcgat agcctgcacg agcacattgc caatctggcc ggcagccccg ccattaagaa 4081 gggcatcctg cagacagtga aggtggtgga cgagctcgtg aaagtgatgg gccggcacaa 4141 gcccgagaac atcgtgatcg aaatggccag agagaaccag accacccaga agggacagaa 4201 gaacagccgc gagagaatga agcggatcga agagggcatc aaagagctgg gcagccagat 4261 cctgaaagaa caccccgtgg aaaacaccca gctgcagaac gagaagctgt acctgtacta 4321 cctgcagaat gggcgggata tgtacgtgga ccaggaactg gacatcaacc ggctgtccga 4381 ctacgatgtg gaccatatcg tgcctcagag ctttctgaag gacgactcca tcgacaacaa 4441 ggtgctgacc agaagcgaca agaaccgggg caagagcgac aacgtgccct ccgaagaggt 4501 cgtgaagaag atgaagaact actggcggca gctgctgaac gccaagctga ttacccagag 4561 aaagttcgac aatctgacca aggccgagag aggcggcctg agcgaactgg ataaggccgg 4621 cttcatcaag agacagctgg tggaaacccg gcagatcaca aagcacgtgg cacagatcct 4681 ggactcccgg atgaacacta agtacgacga gaatgacaag ctgatccggg aagtgaaagt 4741 gatcaccctg aagtccaagc tggtgtccga tttccggaag gatttccagt tttacaaagt 4801 gcgcgagatc aacaactacc accacgccca cgacgcctac ctgaacgccg tcgtgggaac 4861 cgccctgatc aaaaagtacc ctaagctgga aagcgagttc gtgtacggcg actacaaggt 4921 gtacgacgtg cggaagatga tcgccaagag cgagcaggaa atcggcaagg ctaccgccaa 4981 gtacttcttc tacagcaaca tcatgaactt tttcaagacc gagattaccc tggccaacgg 5041 cgagatccgg aagcggcctc tgatcgagac aaacggcgaa accggggaga tcgtgtggga 5101 taagggccgg gattttgcca ccgtgcggaa agtgctgagc atgccccaag tgaatatcgt 5161 gaaaaagacc gaggtgcaga caggcggctt cagcaaagag tctatcctgc ccaagaggaa 5221 cagcgataag ctgatcgcca gaaagaagga ctgggaccct aagaagtacg gcggcttcga 5281 cagccccacc gtggcctatt ctgtgctggt ggtggccaaa gtggaaaagg gcaagtccaa 5341 gaaactgaag agtgtgaaag agctgctggg gatcaccatc atggaaagaa gcagcttcga 5401 gaagaatccc atcgactttc tggaagccaa gggctacaaa gaagtgaaaa aggacctgat 5461 catcaagctg cctaagtact ccctgttcga gctggaaaac ggccggaaga gaatgctggc 5521 ctctgccggc gaactgcaga agggaaacga actggccctg ccctccaaat atgtgaactt 5581 cctgtacctg gccagccact atgagaagct gaagggctcc cccgaggata atgagcagaa 5641 acagctgttt gtggaacagc acaagcacta cctggacgag atcatcgagc agatcagcga 5701 gttctccaag agagtgatcc tggccgacgc taatctggac aaagtgctgt ccgcctacaa 5761 caagcaccgg gataagccca tcagagagca ggccgagaat atcatccacc tgtttaccct 5821 gaccaatctg ggagcccctg ccgccttcaa gtactttgac accaccatcg accggaagag 5881 gtacaccagc accaaagagg tgctggacgc caccctgatc caccagagca tcaccggcct 5941 gtacgagaca cggatcgacc tgtctcagct gggaggcgac aaaaggccgg cggccacgaa 6001 aaaggccggc caggcaaaaa agaaaaagta agaattcctc gagcaccacc accaccacca 6061 ctgagaattc tgtattacgc cgttttaaga gctccttcga cgcctttttg tccttcagct 6121 gctccacggt gactcgcttc tctctctttc cacagtgtct cttttttctt ttcactctct 6181 atacaaatgt gagcgacctc cttttctgta caacggcctt ccggcgtgtg cttttctcat 6241 cgacctctcc tcgcttcttg ggcactcctt catcgagcaa acaagacgag ggaggtgacg 6301 cgattggtga gaccactacg agtatggacg gagctgtctg ataccccctg ttttttatct 6361 tgtaccccgc tctgcagtga cggtagcccg ctgctgtgct tcggtatcgc cgcttcatac 6421 gcttctctct tttttcaacg tgcggcgctg attcaaaggt tacctcaacg cgacccgccg 6481 ctgcatcctt tgttgctcct cttgtcgcaa acaaacaaaa aacgtgctgt gctttttcct 6541 ttatcgtgtc tttttgcaaa gtctagagtt taccgacaag accagaatag cgcttatgtg 6601 tgtgtgtttg tgtgtgggtg tgtgtgccca tgtgtgacaa agaggccagt gctttctcta 6661 aagcaagagc gcgaggttgc tggctaagga ctctaagtcc ctcacagtga tggcgtgttg 6721 cattttctaa accgtcaatc caacacgtcg gtagcatatc ggttttgtag gctcttctgg 6781 ataccttccc cagtaagacc gccactgctt cgtgactgaa gcactgtaaa tatcactgaa 6841 accgtcgcgt tgctttgtct aattatcacc gcctttgcca gctatcgcct caggctgcgc 6901 tctttgacta atccactcac cacttctctg ccttcttcct ccggttgttg ttgtgtcctg 6961 cgtgtacatg gcgcgtgtcc ttttcgagca aacagctgtc ttttctttca cgataacaca 7021 ctcatattaa acgcgagtat tactcatcag tcaacgtcac attccgctct gtccacttcg 7081 accttacacc tctacttgtc caactatctt ccacttgtca agcgtcgacg taatgaaaaa 7141 gcctgaactc accgcgacgt ctgtcgagaa gtttctgatc gaaaagttcg acagcgtctc 7201 cgacctgatg cagctctcgg agggcgaaga atctcgtgct ttcagcttcg atgtaggagg 7261 gcgtggatat gtcctgcggg taaatagctg cgccgatggt ttctacaaag atcgttatgt 7321 ttatcggcac tttgcatcgg ccgcgctccc gattccggaa gtgcttgaca ttggggaatt 7381 tagcgagagc ctgacctatt gcatctcccg ccgtgcacag ggtgtcacgt tgcaagacct 7441 gcctgaaacc gaactgcccg ctgttctgca gccggtcgcg gaggcaatgg atgcgatcgc 7501 tgcggccgat cttagccaga cgagcgggtt cggcccattc ggaccgcaag gaatcggtca 7561 atacactaca tggcgtgatt ttatatgcgc gattgctgat ccccatgtgt atcactggca 7621 aactgtgatg gacgacaccg tcagtgcgtc cgtcgcgcag gctctcgatg agctgatgct 7681 ttgggccgag gactgccccg aagtccggca cctcgtgcac gcggatttcg gctccaacaa 7741 tgtcctgacg gacaatggcc gcataacagc ggtcattgac tggagcgagg cgatgttcgg 7801 ggattcccaa tacgaggtcg ccaacatctt cttctggagg ccgtggttgg cttgtatgga 7861 gcagcagacg cgctacttcg agcggaggca tccggagctt gcaggatcgc ctcggctccg 7921 ggcgtatatg ctccgcattg gtcttgacca actctatcag agcttggttg acggcaattt 7981 cgatgatgca gcttgggcgc agggtcgatg cgacgcaatc gtccgatccg gagccgggac 8041 tgtcgggcgt acacaaatcg cccgcagaag cgcggccgtc tggaccgatg gctgtgtaga 8101 agtactcgcc gatagtggaa accgacgccc cagcactcgt ccgagggcaa aggaatagta 8161 ggatcccgta tctcgaggat ctagaggatc cagcaggcgg agaaagaggg aagtataagg 8221 cggacgcata ggtcggagta tcgagaaaaa gaggcagaga tgggtggtgg cggagcgccc 8281 ctctctgccc ttctctgttt tactgttccc gccacgtcgc caactcctct ttttgtttgc 8341 tctaatgtcg gtgctatcct cttcctcttt ttactctccg tttttcttcc ttccgttttg 8401 tcttgttatc accgttattt ttcttcttct ttcttagccg atttgggtct cctgcctacg 8461 gcagcggtga tgagtcgcac ttccgtcccc ctttcctctc cgtaagtact ccctcgatgc 8521 ctcaggcgct tctattttgc ggcactgtgc tgaccacctc ccacgtgtgc agtgagagcg 8581 ccagagacat tcaggagaag agggaagagg ggaagtaaat accaaagcga ggaagatgtc 8641 tttctcgctg cttctcatcc tgttgaccgg tgtgcacggc ggtgtgtcct ctcggcttcc 8701 ctcctccttc cctcgctccc ctttttctgt gttttttctt tctaacatga ttgcgcctgc 8761 tctttttgcc ccgcagcgtc gcagtgggtg tcacctgcga ccgccgtacg ttttactata 8821 tacatatttg tttctgtgct gatgccattt gtccgctact ccatatcgtg cgtgtttgtt 8881 ttccagtggt gacgccttcc gcatcatttt aatcaattcg aggcgaaaag ggtgatttga 8941 gtgttggagt gggctttttt atttatttga ctccaaacat cttttctttt cccggtatct 9001 tcgggtccgt cacgcaggtc ggtgggtgtg tccgagtgcg cttctttact cacgtgccgc 9061 tgcgcacata cacagatttt aaaagcacgc atacacgatc tgtgccttca aaactaatca 9121 acaaaaacaa aaatacaaaa aagacaagaa taagaggtca gcaacgcacc cacggctcct 9181 ttcttcctga tccacgtcgt ggccgctgta cgctctcaaa cacgctttgg cgctgatgcg 9241 tgcctttata caaagaacaa agaagaagcg aaaaacggga gcgtgttgct ttggggatgt 9301 atgcgtgcca cagttctgcg gtacaattcg taatcatggt catagctgtt tcctgtgtga 9361 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaatgc 9421 atgcggccgc acagtgtgtg ggtgaggtgc gcgaaggtgt gtctgtcggt gggggagctc 9481 gcgcggtgtg tgcgttggcg gctcagcggc ccccctcgcg tctctcacgc cggcccgcct 9541 gtccccctcc cccacataaa aggacgcagc gcgctcagct tttgtgtgca tgtatgctat 9601 cgctggtcgc gtgtgctgcg cctctgtatc ttcttttgaa tctttccctt tttttcgctt 9661 tcttttatct tcctcccgtg taccgcagtt gatggatcag gcgccctgtt gcaacttggg 9721 ccgccgacgg tgctggtgcg aggcagcagg ccagcatgtt gccgagatct ttaattaaaa 9781 tccctagcct gcaggggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt 9841 cacaccgcat acgtcaaagc aaccatagta cgcgccctgt agcggcgcat taagcgcggc 9901 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc 9961 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa 10021 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact 10081 tgatttgggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt 10141 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa 10201 ccctatctcg ggctattctt ttgatttata agggattttg ccgatttcgg cctattggtt 10261 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac 10321 aattttatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg 10381 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta 10441 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc 10501 gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat 10561 aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat 10621 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata 10681 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct 10741 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa 10801 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa 10861 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt 10921 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg 10981 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca 11041 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa 11101 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt 11161 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc 11221 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa 11281 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga 11341 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc 11401 tgataaatct ggagccggtg agcgtggaag ccgcggtatc attgcagcac tggggccaga 11461 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga 11521 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aa // LOCUS pTadd 6658 bp ds-DNA circular SYN 12-SEP-2016 DEFINITION pTadd ACCESSION . VERSION . KEYWORDS pTadd SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6658) AUTHORS . TITLE Direct Submission JOURNAL Exported Friday, Oct 21, 2016 from SnapGene Viewer 3.2.1 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6658 /organism="synthetic DNA construct" /mol_type="other DNA" rep_origin 171..759 /direction=RIGHT /note="ori" /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" misc_feature 841..1184 /note="5'target beta" misc_feature 1185..1741 /note="PGKB5'" misc_feature 1742..1809 /note="Addback goes here" misc_feature 1810..2301 /note="CfPGKA3'" misc_feature 2302..2863 /note="CfPGKB5'" CDS 2869..3243 /codon_start=1 /gene="Sh ble from Streptoalloteichus hindustanus" /product="antibiotic-binding protein" /note="BleoR" /note="confers resistance to bleomycin, phleomycin, and Zeocin(TM)" /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR EFALRDPAGNCVHFVAEEQD" misc_feature 3273..4502 /note="CfGSPS 3'" primer_bind complement(4425..4441) /note="M13 rev" /note="common sequencing primer, one of multiple similar variants" protein_bind 4449..4465 /bound_moiety="lac repressor encoded by lacI" /note="lac operator" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." promoter complement(4473..4503) /note="lac promoter" /note="promoter for the E. coli lac operon" misc_feature 4517..4850 /note="3'target beta" rep_origin 4956..5411 /direction=RIGHT /note="f1 ori" /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" promoter 5693..5797 /gene="bla" /note="AmpR promoter" CDS 5798..6658 /codon_start=1 /gene="bla" /product="beta-lactamase" /note="AmpR" /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" ORIGIN 1 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt 61 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag 121 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct 181 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt 241 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg 301 cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct 361 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc 421 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg 481 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa 541 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg 601 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg 661 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga 721 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt 781 ttacggttcc tggccttttg ctggcctttt gctcacatgt aagcttttaa ttaaggtacc 841 caaagaaacg gcgcttcctc gtccgcatgt gtttgcggga catgacgccg cgccactgcc 901 acgccttttt cttgcctgct gtgaggagcg gccgcggccc ttggcgctcc cccccccgcc 961 tgcgtctccg ccagcggcgg ccacctgtgt cacaccccat ctcccccgct ctctctcccc 1021 cgtctctcct ggtctcgaaa caacccgaca ttcggaaata tgtttgcacg gcgttgcgcg 1081 cgtgtcacac acgcacacac acaccgacca gtaaccgtcc cccaacaccc cctcctccgc 1141 acaaagcata ccctttcgtt tcgcctttcg ccactctgcc atcctcgaga ccgacaagac 1201 cagaatagcg cttatgtgtg tgtgtttgtg tgtgggtgtg tgtgcccatg tgtgacaaag 1261 aggccagtgc tttctctaaa gcaagagcgc gaggttgctg gctaaggact ctaagtccct 1321 cacagtgatg gcgtgttgca ttttctaaac cgtcaatcca acacgtcggt agcatatcgg 1381 ttttgtaggc tcttctggat accttcccca gtaagaccgc cactgcttcg tgactgaagc 1441 actgtaaata tcactgaaac cgtcgcgttg ctttgtctaa ttatcaccgc ctttgccagc 1501 tatcgcctca ggctgcgctc tttgactaat ccactcacca cttctctgcc ttcttcctcc 1561 ggttgttgtt gtgtcctgcg tgtacatggc gcgtgtcctt ttcgagcaaa cagctgtctt 1621 ttctttcacg ataacacact catattaaac gcgagtatta ctcatcagtc aacgtcacat 1681 tccgctctgt ccacttcgac cttacacctc tacttgtcca actatcttcc acttgtcaag 1741 cactagtctt aaggtaacta tcgatttagc tcaattgcca tggcctgatc ctcgagcggc 1801 tatgaattct gtattacgcc gttttaagag ctccttcgac gcctttttgt ccttcagctg 1861 ctccacggtg actcgcttct ctctctttcc acagtgtctc ttttttcttt tcactctcta 1921 tacaaatgtg agcgacctcc ttttctgtac aacggccttc cggcgtgtgc ttttctcatc 1981 gacctctcct cgcttcttgg gcactccttc atcgagcaaa caagacgagg gaggtgacgc 2041 gattggtgag accactacga gtatggacgg agctgtctga taccccctgt tttttatctt 2101 gtaccccgct ctgcagtgac ggtagcccgc tgctgtgctt cggtatcgcc gcttcatacg 2161 cttctctctt ttttcaacgt gcggcgctga ttcaaaggtt acctcaacgc gacccgccgc 2221 tgcatccttt gttgctcctc ttgtcgcaaa caaacaaaaa acgtgctgtg ctttttcctt 2281 tatcgtgtct ttttgcaaag tctagagttt accgacaaga ccagaatagc gcttatgtgt 2341 gtgtgtttgt gtgtgggtgt gtgtgcccat gtgtgacaaa gaggccagtg ctttctctaa 2401 agcaagagcg cgaggttgct ggctaaggac tctaagtccc tcacagtgat ggcgtgttgc 2461 attttctaaa ccgtcaatcc aacacgtcgg tagcatatcg gttttgtagg ctcttctgga 2521 taccttcccc agtaagaccg ccactgcttc gtgactgaag cactgtaaat atcactgaaa 2581 ccgtcgcgtt gctttgtcta attatcaccg cctttgccag ctatcgcctc aggctgcgct 2641 ctttgactaa tccactcacc acttctctgc cttcttcctc cggttgttgt tgtgtcctgc 2701 gtgtacatgg cgcgtgtcct tttcgagcaa acagctgtct tttctttcac gataacacac 2761 tcatattaaa cgcgagtatt actcatcagt caacgtcaca ttccgctctg tccacttcga 2821 ccttacacct ctacttgtcc aactatcttc cacttgtcaa gcgtcgacat ggccaagttg 2881 accagtgccg ttccggtgct caccgcgcgc gacgtcgccg gagcggtcga gttctggacc 2941 gaccggctcg ggttctcccg ggacttcgtg gaggacgact tcgccggtgt ggtccgggac 3001 gacgtgaccc tgttcatcag cgcggtccag gaccaggtgg tgccggacaa caccctggcc 3061 tgggtgtggg tgcgcggcct ggacgagctg tacgccgagt ggtcggaggt cgtgtccacg 3121 aacttccggg acgcctccgg gccggccatg accgagatcg gcgagcagcc gtgggggcgg 3181 gagttcgccc tgcgcgaccc ggccggcaac tgcgtgcact tcgtggccga ggagcaggac 3241 tgaggaggat cccgtatctc gaggatctag aggatccagc aggcggagaa agagggaagt 3301 ataaggcgga cgcataggtc ggagtatcga gaaaaagagg cagagatggg tggtggcgga 3361 gcgcccctct ctgcccttct ctgttttact gttcccgcca cgtcgccaac tcctcttttt 3421 gtttgctcta atgtcggtgc tatcctcttc ctctttttac tctccgtttt tcttccttcc 3481 gttttgtctt gttatcaccg ttatttttct tcttctttct tagccgattt gggtctcctg 3541 cctacggcag cggtgatgag tcgcacttcc gtcccccttt cctctccgta agtactccct 3601 cgatgcctca ggcgcttcta ttttgcggca ctgtgctgac cacctcccac gtgtgcagtg 3661 agagcgccag agacattcag gagaagaggg aagaggggaa gtaaatacca aagcgaggaa 3721 gatgtctttc tcgctgcttc tcatcctgtt gaccggtgtg cacggcggtg tgtcctctcg 3781 gcttccctcc tccttccctc gctccccttt ttctgtgttt tttctttcta acatgattgc 3841 gcctgctctt tttgccccgc agcgtcgcag tgggtgtcac ctgcgaccgc cgtacgtttt 3901 actatataca tatttgtttc tgtgctgatg ccatttgtcc gctactccat atcgtgcgtg 3961 tttgttttcc agtggtgacg ccttccgcat cattttaatc aattcgaggc gaaaagggtg 4021 atttgagtgt tggagtgggc ttttttattt atttgactcc aaacatcttt tcttttcccg 4081 gtatcttcgg gtccgtcacg caggtcggtg ggtgtgtccg agtgcgcttc tttactcacg 4141 tgccgctgcg cacatacaca gattttaaaa gcacgcatac acgatctgtg ccttcaaaac 4201 taatcaacaa aaacaaaaat acaaaaaaga caagaataag aggtcagcaa cgcacccacg 4261 gctcctttct tcctgatcca cgtcgtggcc gctgtacgct ctcaaacacg ctttggcgct 4321 gatgcgtgcc tttatacaaa gaacaaagaa gaagcgaaaa acgggagcgt gttgctttgg 4381 ggatgtatgc gtgccacagt tctgcggtac aattcgtaat catggtcata gctgtttcct 4441 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt 4501 aaatgcatgc ggccgcacag tgtgtgggtg aggtgcgcga aggtgtgtct gtcggtgggg 4561 gagctcgcgc ggtgtgtgcg ttggcggctc agcggccccc ctcgcgtctc tcacgccggc 4621 ccgcctgtcc ccctccccca cataaaagga cgcagcgcgc tcagcttttg tgtgcatgta 4681 tgctatcgct ggtcgcgtgt gctgcgcctc tgtatcttct tttgaatctt tccctttttt 4741 tcgctttctt ttatcttcct cccgtgtacc gcagttgatg gatcaggcgc cctgttgcaa 4801 cttgggccgc cgacggtgct ggtgcgaggc agcaggccag catgttgccg agatctttaa 4861 ttaaaatccc tagcctgcag gggcgcctga tgcggtattt tctccttacg catctgtgcg 4921 gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg gcgcattaag 4981 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc 5041 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc 5101 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa 5161 aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg 5221 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac 5281 actcaaccct atctcgggct attcttttga tttataaggg attttgccga tttcggccta 5341 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac 5401 gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca 5461 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc 5521 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc 5581 atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt 5641 catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac 5701 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc 5761 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt 5821 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct 5881 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga 5941 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag 6001 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca 6061 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga 6121 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag 6181 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc 6241 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa 6301 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt 6361 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg 6421 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt 6481 tattgctgat aaatctggag ccggtgagcg tggaagccgc ggtatcattg cagcactggg 6541 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat 6601 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaa // LOCUS pTB007_Cas9_T7_Tub 15446 bp ds-DNA circular SYN 21-OCT-2016 DEFINITION pTB007_Cas9_T7_Tub ACCESSION . VERSION . KEYWORDS pT007_Cas9_T7_Tub SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 15446) AUTHORS . TITLE Direct Submission JOURNAL Exported Friday, Oct 21, 2016 from SnapGene Viewer 3.2.1 http://www.snapgene.com FEATURES Location/Qualifiers source 1..15446 /organism="synthetic DNA construct" /mol_type="other DNA" rep_origin 171..759 /direction=RIGHT /note="ori" /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" misc_feature 841..1184 /note="5'target beta tub" misc_feature 1185..1741 /note="PGKB5'" misc_feature 1423..2239 /note="sequenced 11/5" misc_feature 1565..2239 /note="sequenced 11/2" CDS 1763..1828 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /note="3xFLAG" /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 1835..1855 /codon_start=1 /product="nuclear localization signal of SV40 large T antigen" /note="SV40 NLS" /translation="PKKKRKV" CDS 1880..5980 /codon_start=1 /product="Cas9 (Csn1) endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /note="Cas9" /note="generates RNA-guided double strand breaks in DNA" /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGD" CDS 5981..6028 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /note="nucleoplasmin NLS" /translation="KRPAATKKAGQAKKKK" misc_feature 6039..6562 /note="CfPGKA 3'" CDS 6044..6061 /codon_start=1 /product="6xHis affinity tag" /note="6xHis" /translation="HHHHHH" misc_feature 6568..6956 /note="Calmodulin IR" CDS 6957..9644 /codon_start=1 /note="T7 RNAP" /translation="METINIAKNEFLEPPKKKRKVEFSDIELAAIPFNTLADHYGERLA REQLALEHESYEMGEARFRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEV KAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARF GRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSI HVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQ PCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAW KINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRK DKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTL AKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQ DSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPS ETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAY GVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIW ESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKK PIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHE KYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLD KMPALPAKGNLNLRDILESDFAFA" CDS 6999..7019 /codon_start=1 /product="nuclear localization signal of SV40 large T antigen" /note="SV40 NLS" /translation="PKKKRKV" misc_feature 7198..7762 /note="sequenced 11/5" misc_feature 7274..7707 /note="sequenced 11/2" misc_feature 9645..10997 /note="Calmodulin IR" misc_feature 10585..11222 /note="sequenced 11/2" misc_feature 10585..11180 /note="sequenced 11/5" CDS 11007..12032 /codon_start=1 /gene="aph(4)-Ia" /product="aminoglycoside phosphotransferase from E. coli" /note="HygR" /note="confers resistance to hygromycin" /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK E" misc_feature 12061..13290 /note="CfPGKA 3'" primer_bind complement(13213..13229) /note="M13 rev" /note="common sequencing primer, one of multiple similar variants" protein_bind 13237..13253 /bound_moiety="lac repressor encoded by lacI" /note="lac operator" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." promoter complement(13261..13291) /note="lac promoter" /note="promoter for the E. coli lac operon" misc_feature 13292..13649 /note="3' beta tub" rep_origin 13744..14199 /direction=RIGHT /note="f1 ori" /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" promoter 14481..14585 /gene="bla" /note="AmpR promoter" CDS 14586..15446 /codon_start=1 /gene="bla" /product="beta-lactamase" /note="AmpR" /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" ORIGIN 1 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt 61 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag 121 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct 181 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt 241 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg 301 cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct 361 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc 421 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg 481 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa 541 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg 601 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg 661 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga 721 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt 781 ttacggttcc tggccttttg ctggcctttt gctcacatgt aagcttttaa ttaaggtacc 841 caaagaaacg gcgcttcctc gtccgcatgt gtttgcggga catgacgccg cgccactgcc 901 acgccttttt cttgcctgct gtgaggagcg gccgcggccc ttggcgctcc cccccccgcc 961 tgcgtctccg ccagcggcgg ccacctgtgt cacaccccat ctcccccgct ctctctcccc 1021 cgtctctcct ggtctcgaaa caacccgaca ttcggaaata tgtttgcacg gcgttgcgcg 1081 cgtgtcacac acgcacacac acaccgacca gtaaccgtcc cccaacaccc cctcctccgc 1141 acaaagcata ccctttcgtt tcgcctttcg ccactctgcc atcctcgaga ccgacaagac 1201 cagaatagcg cttatgtgtg tgtgtttgtg tgtgggtgtg tgtgcccatg tgtgacaaag 1261 aggccagtgc tttctctaaa gcaagagcgc gaggttgctg gctaaggact ctaagtccct 1321 cacagtgatg gcgtgttgca ttttctaaac cgtcaatcca acacgtcggt agcatatcgg 1381 ttttgtaggc tcttctggat accttcccca gtaagaccgc cactgcttcg tgactgaagc 1441 actgtaaata tcactgaaac cgtcgcgttg ctttgtctaa ttatcaccgc ctttgccagc 1501 tatcgcctca ggctgcgctc tttgactaat ccactcacca cttctctgcc ttcttcctcc 1561 ggttgttgtt gtgtcctgcg tgtacatggc gcgtgtcctt ttcgagcaaa cagctgtctt 1621 ttctttcacg ataacacact catattaaac gcgagtatta ctcatcagtc aacgtcacat 1681 tccgctctgt ccacttcgac cttacacctc tacttgtcca actatcttcc acttgtcaag 1741 cactagtacc ggtgccacca tggactataa ggaccacgac ggagactaca aggatcatga 1801 tattgattac aaagacgatg acgataagat ggccccaaag aagaagcgga aggtcggtat 1861 ccacggagtc ccagcagccg acaagaagta cagcatcggc ctggacatcg gcaccaactc 1921 tgtgggctgg gccgtgatca ccgacgagta caaggtgccc agcaagaaat tcaaggtgct 1981 gggcaacacc gaccggcaca gcatcaagaa gaacctgatc ggagccctgc tgttcgacag 2041 cggcgaaaca gccgaggcca cccggctgaa gagaaccgcc agaagaagat acaccagacg 2101 gaagaaccgg atctgctatc tgcaagagat cttcagcaac gagatggcca aggtggacga 2161 cagcttcttc cacagactgg aagagtcctt cctggtggaa gaggataaga agcacgagcg 2221 gcaccccatc ttcggcaaca tcgtggacga ggtggcctac cacgagaagt accccaccat 2281 ctaccacctg agaaagaaac tggtggacag caccgacaag gccgacctgc ggctgatcta 2341 tctggccctg gcccacatga tcaagttccg gggccacttc ctgatcgagg gcgacctgaa 2401 ccccgacaac agcgacgtgg acaagctgtt catccagctg gtgcagacct acaaccagct 2461 gttcgaggaa aaccccatca acgccagcgg cgtggacgcc aaggccatcc tgtctgccag 2521 actgagcaag agcagacggc tggaaaatct gatcgcccag ctgcccggcg agaagaagaa 2581 tggcctgttc ggaaacctga ttgccctgag cctgggcctg acccccaact tcaagagcaa 2641 cttcgacctg gccgaggatg ccaaactgca gctgagcaag gacacctacg acgacgacct 2701 ggacaacctg ctggcccaga tcggcgacca gtacgccgac ctgtttctgg ccgccaagaa 2761 cctgtccgac gccatcctgc tgagcgacat cctgagagtg aacaccgaga tcaccaaggc 2821 ccccctgagc gcctctatga tcaagagata cgacgagcac caccaggacc tgaccctgct 2881 gaaagctctc gtgcggcagc agctgcctga gaagtacaaa gagattttct tcgaccagag 2941 caagaacggc tacgccggct acattgacgg cggagccagc caggaagagt tctacaagtt 3001 catcaagccc atcctggaaa agatggacgg caccgaggaa ctgctcgtga agctgaacag 3061 agaggacctg ctgcggaagc agcggacctt cgacaacggc agcatccccc accagatcca 3121 cctgggagag ctgcacgcca ttctgcggcg gcaggaagat ttttacccat tcctgaagga 3181 caaccgggaa aagatcgaga agatcctgac cttccgcatc ccctactacg tgggccctct 3241 ggccagggga aacagcagat tcgcctggat gaccagaaag agcgaggaaa ccatcacccc 3301 ctggaacttc gaggaagtgg tggacaaggg cgcttccgcc cagagcttca tcgagcggat 3361 gaccaacttc gataagaacc tgcccaacga gaaggtgctg cccaagcaca gcctgctgta 3421 cgagtacttc accgtgtata acgagctgac caaagtgaaa tacgtgaccg agggaatgag 3481 aaagcccgcc ttcctgagcg gcgagcagaa aaaggccatc gtggacctgc tgttcaagac 3541 caaccggaaa gtgaccgtga agcagctgaa agaggactac ttcaagaaaa tcgagtgctt 3601 cgactccgtg gaaatctccg gcgtggaaga tcggttcaac gcctccctgg gcacatacca 3661 cgatctgctg aaaattatca aggacaagga cttcctggac aatgaggaaa acgaggacat 3721 tctggaagat atcgtgctga ccctgacact gtttgaggac agagagatga tcgaggaacg 3781 gctgaaaacc tatgcccacc tgttcgacga caaagtgatg aagcagctga agcggcggag 3841 atacaccggc tggggcaggc tgagccggaa gctgatcaac ggcatccggg acaagcagtc 3901 cggcaagaca atcctggatt tcctgaagtc cgacggcttc gccaacagaa acttcatgca 3961 gctgatccac gacgacagcc tgacctttaa agaggacatc cagaaagccc aggtgtccgg 4021 ccagggcgat agcctgcacg agcacattgc caatctggcc ggcagccccg ccattaagaa 4081 gggcatcctg cagacagtga aggtggtgga cgagctcgtg aaagtgatgg gccggcacaa 4141 gcccgagaac atcgtgatcg aaatggccag agagaaccag accacccaga agggacagaa 4201 gaacagccgc gagagaatga agcggatcga agagggcatc aaagagctgg gcagccagat 4261 cctgaaagaa caccccgtgg aaaacaccca gctgcagaac gagaagctgt acctgtacta 4321 cctgcagaat gggcgggata tgtacgtgga ccaggaactg gacatcaacc ggctgtccga 4381 ctacgatgtg gaccatatcg tgcctcagag ctttctgaag gacgactcca tcgacaacaa 4441 ggtgctgacc agaagcgaca agaaccgggg caagagcgac aacgtgccct ccgaagaggt 4501 cgtgaagaag atgaagaact actggcggca gctgctgaac gccaagctga ttacccagag 4561 aaagttcgac aatctgacca aggccgagag aggcggcctg agcgaactgg ataaggccgg 4621 cttcatcaag agacagctgg tggaaacccg gcagatcaca aagcacgtgg cacagatcct 4681 ggactcccgg atgaacacta agtacgacga gaatgacaag ctgatccggg aagtgaaagt 4741 gatcaccctg aagtccaagc tggtgtccga tttccggaag gatttccagt tttacaaagt 4801 gcgcgagatc aacaactacc accacgccca cgacgcctac ctgaacgccg tcgtgggaac 4861 cgccctgatc aaaaagtacc ctaagctgga aagcgagttc gtgtacggcg actacaaggt 4921 gtacgacgtg cggaagatga tcgccaagag cgagcaggaa atcggcaagg ctaccgccaa 4981 gtacttcttc tacagcaaca tcatgaactt tttcaagacc gagattaccc tggccaacgg 5041 cgagatccgg aagcggcctc tgatcgagac aaacggcgaa accggggaga tcgtgtggga 5101 taagggccgg gattttgcca ccgtgcggaa agtgctgagc atgccccaag tgaatatcgt 5161 gaaaaagacc gaggtgcaga caggcggctt cagcaaagag tctatcctgc ccaagaggaa 5221 cagcgataag ctgatcgcca gaaagaagga ctgggaccct aagaagtacg gcggcttcga 5281 cagccccacc gtggcctatt ctgtgctggt ggtggccaaa gtggaaaagg gcaagtccaa 5341 gaaactgaag agtgtgaaag agctgctggg gatcaccatc atggaaagaa gcagcttcga 5401 gaagaatccc atcgactttc tggaagccaa gggctacaaa gaagtgaaaa aggacctgat 5461 catcaagctg cctaagtact ccctgttcga gctggaaaac ggccggaaga gaatgctggc 5521 ctctgccggc gaactgcaga agggaaacga actggccctg ccctccaaat atgtgaactt 5581 cctgtacctg gccagccact atgagaagct gaagggctcc cccgaggata atgagcagaa 5641 acagctgttt gtggaacagc acaagcacta cctggacgag atcatcgagc agatcagcga 5701 gttctccaag agagtgatcc tggccgacgc taatctggac aaagtgctgt ccgcctacaa 5761 caagcaccgg gataagccca tcagagagca ggccgagaat atcatccacc tgtttaccct 5821 gaccaatctg ggagcccctg ccgccttcaa gtactttgac accaccatcg accggaagag 5881 gtacaccagc accaaagagg tgctggacgc caccctgatc caccagagca tcaccggcct 5941 gtacgagaca cggatcgacc tgtctcagct gggaggcgac aaaaggccgg cggccacgaa 6001 aaaggccggc caggcaaaaa agaaaaagta agaattcctc gagcaccacc accaccacca 6061 ctgagaattc tgtattacgc cgttttaaga gctccttcga cgcctttttg tccttcagct 6121 gctccacggt gactcgcttc tctctctttc cacagtgtct cttttttctt ttcactctct 6181 atacaaatgt gagcgacctc cttttctgta caacggcctt ccggcgtgtg cttttctcat 6241 cgacctctcc tcgcttcttg ggcactcctt catcgagcaa acaagacgag ggaggtgacg 6301 cgattggtga gaccactacg agtatggacg gagctgtctg ataccccctg ttttttatct 6361 tgtaccccgc tctgcagtga cggtagcccg ctgctgtgct tcggtatcgc cgcttcatac 6421 gcttctctct tttttcaacg tgcggcgctg attcaaaggt tacctcaacg cgacccgccg 6481 ctgcatcctt tgttgctcct cttgtcgcaa acaaacaaaa aacgtgctgt gctttttcct 6541 ttatcgtgtc tttttgcaaa gtctagacga cacacgaagc gtgtgatgaa tgaaaaggag 6601 gagacactcg acacttgaat atttctccct atcaagccct ttcaggcctg cgtgctccct 6661 caaccccttt ctcgataacg tgcgacgtaa tgttacatgc acacacgtcg cacatctcct 6721 gtgaatggca ctgccttctc gtgccgtcac catgcccgcc tttcagcact ggctcttcgt 6781 ttcttcaccg cttcttcgtt ctgtgaaccc gcgcgcacgc acgcatcgaa gggattcatc 6841 gccattgccc tctccgcctc tctcgctcgc tgcacattga ctctccacag ccccccctcc 6901 ctcgcacagc cagccccacc cacccaccca ccgctctaca cacctccaca gcagccatgg 6961 agacgattaa catcgctaag aacgaattcc tcgagcctcc aaaaaagaag agaaaggtcg 7021 aattctctga catcgaactg gctgctatcc cgttcaacac tctggctgac cattacggtg 7081 agcgtttagc tcgcgaacag ttggcccttg agcatgagtc ttacgagatg ggtgaagcac 7141 gcttccgcaa gatgtttgag cgtcaactta aagctggtga ggttgcggat aacgctgccg 7201 ccaagcctct catcactacc ctactcccta agatgattgc acgcatcaac gactggtttg 7261 aggaagtgaa agctaagcgc ggcaagcgcc cgacagcctt ccagttcctg caagaaatca 7321 agccggaagc cgtagcgtac atcaccatta agaccactct ggcttgccta accagtgctg 7381 acaatacaac cgttcaggct gtagcaagcg caatcggtcg ggccattgag gacgaggctc 7441 gcttcggtcg tatccgtgac cttgaagcta agcacttcaa gaaaaacgtt gaggaacaac 7501 tcaacaagcg cgtagggcac gtctacaaga aagcatttat gcaagttgtc gaggctgaca 7561 tgctctctaa gggtctactc ggtggcgagg cgtggtcttc gtggcataag gaagactcta 7621 ttcatgtagg agtacgctgc atcgagatgc tcattgagtc aaccggaatg gttagcttac 7681 accgccaaaa tgctggcgta gtaggtcaag actctgagac tatcgaactc gcacctgaat 7741 acgctgaggc tatcgcaacc cgtgcaggtg cgctggctgg catctctccg atgttccaac 7801 cttgcgtagt tcctcctaag ccgtggactg gcattactgg tggtggctat tgggctaacg 7861 gtcgtcgtcc tctggcgctg gtgcgtactc acagtaagaa agcactgatg cgctacgaag 7921 acgtttacat gcctgaggtg tacaaagcga ttaacattgc gcaaaacacc gcatggaaaa 7981 tcaacaagaa agtcctagcg gtcgccaacg taatcaccaa gtggaagcat tgtccggtcg 8041 aggacatccc tgcgattgag cgtgaagaac tcccgatgaa accggaagac atcgacatga 8101 atcctgaggc tctcaccgcg tggaaacgtg ctgccgctgc tgtgtaccgc aaggacaagg 8161 ctcgcaagtc tcgccgtatc agccttgagt tcatgcttga gcaagccaat aagtttgcta 8221 accataaggc catctggttc ccttacaaca tggactggcg cggtcgtgtt tacgctgtgt 8281 caatgttcaa cccgcaaggt aacgatatga ccaaaggact gcttacgctg gcgaaaggta 8341 aaccaatcgg taaggaaggt tactactggc tgaaaatcca cggtgcaaac tgtgcgggtg 8401 tcgataaggt tccgttccct gagcgcatca agttcattga ggaaaaccac gagaacatca 8461 tggcttgcgc taagtctcca ctggagaaca cttggtgggc tgagcaagat tctccgttct 8521 gcttccttgc gttctgcttt gagtacgctg gggtacagca ccacggcctg agctataact 8581 gctcccttcc gctggcgttt gacgggtctt gctctggcat ccagcacttc tccgcgatgc 8641 tccgagatga ggtaggtggt cgcgcggtta acttgcttcc tagtgaaacc gttcaggaca 8701 tctacgggat tgttgctaag aaagtcaacg agattctaca agcagacgca atcaatggga 8761 ccgataacga agtagttacc gtgaccgatg agaacactgg tgaaatctct gagaaagtca 8821 agctgggcac taaggcactg gctggtcaat ggctggctta cggtgttact cgcagtgtga 8881 ctaagcgttc agtcatgacg ctggcttacg ggtccaaaga gttcggcttc cgtcaacaag 8941 tgctggaaga taccattcag ccagctattg attccggcaa gggtctgatg ttcactcagc 9001 cgaatcaggc tgctggatac atggctaagc tgatttggga atctgtgagc gtgacggtgg 9061 tagctgcggt tgaagcaatg aactggctta agtctgctgc taagctgctg gctgctgagg 9121 tcaaagataa gaagactgga gagattcttc gcaagcgttg cgctgtgcat tgggtaactc 9181 ctgatggttt ccctgtgtgg caggaataca agaagcctat tcagacgcgc ttgaacctga 9241 tgttcctcgg tcagttccgc ttacagccta ccattaacac caacaaagat agcgagattg 9301 atgcacacaa acaggagtct ggtatcgctc ctaactttgt acacagccaa gacggtagcc 9361 accttcgtaa gactgtagtg tgggcacacg agaagtacgg aatcgaatct tttgcactga 9421 ttcacgactc cttcggtacc attccggctg acgctgcgaa cctgttcaaa gcagtgcgcg 9481 aaactatggt tgacacatat gagtcttgtg atgtactggc tgatttctac gaccagttcg 9541 ctgaccagtt gcacgagtct caattggaca aaatgccagc acttccggct aaaggtaact 9601 tgaacctccg tgacatctta gagtcggact tcgcgttcgc gtaggcggcc gccctcctcc 9661 tcctttcttg ttcctttcac gtcgccttct cggttgtagc tggcagacga cgagtcttac 9721 ttttacgtgt acttctctat agatgatgta tgatctctct gcatgcgtgt tcgtgcatgt 9781 gtccgtgtgt tgtgtacgcg tgcgtctcgc ctcagctctc cgcgtgaaag ggtttgactg 9841 cccatgatgc gtgtgtatat ccacgcgcag gcacgcacac acacacacac acacacacac 9901 acaggcacac acaggcacac aaacgcatct caggccgagc cgcatacgtc tctcgcacgg 9961 tctcgtttat ttgatcatgt agttgagtat taaattggga agacaaaaac ataatagcac 10021 gaagagtcgg gcacgaaaag cccgatctct ctctctctct ctcgtgcgcg caggggcgtg 10081 tgggtgcacg acgacgagga cggagggggg aagggaggca taaacggatc ccccatcgca 10141 catgcgggta cgagcattat ctgctgtgtc tggtccttta tcatatcgct acccgcccgc 10201 cccccgcccc cctccccccc ccccccccgc ggctcctgtc actgtcgttc ctgcgactcc 10261 ccacacaccc gcgcaacgct catgtcacga agagaaaagt agaaggcgtg gcgatgcgtt 10321 gcgcggcccc ctgtccgctg gcatgcagac gtcgagccgt ggaacggatg gtagcgagag 10381 acaacgcggc gatgcaggaa aggagattta ctgcaggacg tctacacacg cgcgcacacc 10441 actgcgaatg gcaccgtgct gaggaaaatt gggggggagg gcgcacgggg gcggggcagg 10501 aaacggttgg atcagcagca ctctcaactt gtgtccgtat tgcgaaggag ggagaggagc 10561 ctgcccgtat tatgggcgct gaaacgtcgg aaccacctat gaatcccact tctgcgtcgc 10621 ggaggcgtac tgcttccgac cacctccgca gctcacgcgc gtcgagctcc ctccctttcc 10681 ccttctttcc gttgtgtgtg tgtgtgtgtg tgtgtcttgt cacgatgcat ggccatctct 10741 cctccaaccc tccacgcctt cctctccccc gcccatcaaa cgcgctacgg ccacaaccat 10801 ttcttcaagt atcacatcca ccaccactct tacccaccta ccctcttcga ctctgacaca 10861 gccactgacg ccctctccgc ctctctcgct cgctgcacat tgactctcca cagccccccc 10921 tccctcgcac agccagcccc acccacccac ccacccaccc accgctctac acacctccac 10981 agcagccaga tcctagtgtc gacgtaatga aaaagcctga actcaccgcg acgtctgtcg 11041 agaagtttct gatcgaaaag ttcgacagcg tctccgacct gatgcagctc tcggagggcg 11101 aagaatctcg tgctttcagc ttcgatgtag gagggcgtgg atatgtcctg cgggtaaata 11161 gctgcgccga tggtttctac aaagatcgtt atgtttatcg gcactttgca tcggccgcgc 11221 tcccgattcc ggaagtgctt gacattgggg aatttagcga gagcctgacc tattgcatct 11281 cccgccgtgc acagggtgtc acgttgcaag acctgcctga aaccgaactg cccgctgttc 11341 tgcagccggt cgcggaggca atggatgcga tcgctgcggc cgatcttagc cagacgagcg 11401 ggttcggccc attcggaccg caaggaatcg gtcaatacac tacatggcgt gattttatat 11461 gcgcgattgc tgatccccat gtgtatcact ggcaaactgt gatggacgac accgtcagtg 11521 cgtccgtcgc gcaggctctc gatgagctga tgctttgggc cgaggactgc cccgaagtcc 11581 ggcacctcgt gcacgcggat ttcggctcca acaatgtcct gacggacaat ggccgcataa 11641 cagcggtcat tgactggagc gaggcgatgt tcggggattc ccaatacgag gtcgccaaca 11701 tcttcttctg gaggccgtgg ttggcttgta tggagcagca gacgcgctac ttcgagcgga 11761 ggcatccgga gcttgcagga tcgcctcggc tccgggcgta tatgctccgc attggtcttg 11821 accaactcta tcagagcttg gttgacggca atttcgatga tgcagcttgg gcgcagggtc 11881 gatgcgacgc aatcgtccga tccggagccg ggactgtcgg gcgtacacaa atcgcccgca 11941 gaagcgcggc cgtctggacc gatggctgtg tagaagtact cgccgatagt ggaaaccgac 12001 gccccagcac tcgtccgagg gcaaaggaat agtaggatcc cgtatctcga ggatctagag 12061 gatccagcag gcggagaaag agggaagtat aaggcggacg cataggtcgg agtatcgaga 12121 aaaagaggca gagatgggtg gtggcggagc gcccctctct gcccttctct gttttactgt 12181 tcccgccacg tcgccaactc ctctttttgt ttgctctaat gtcggtgcta tcctcttcct 12241 ctttttactc tccgtttttc ttccttccgt tttgtcttgt tatcaccgtt atttttcttc 12301 ttctttctta gccgatttgg gtctcctgcc tacggcagcg gtgatgagtc gcacttccgt 12361 ccccctttcc tctccgtaag tactccctcg atgcctcagg cgcttctatt ttgcggcact 12421 gtgctgacca cctcccacgt gtgcagtgag agcgccagag acattcagga gaagagggaa 12481 gaggggaagt aaataccaaa gcgaggaaga tgtctttctc gctgcttctc atcctgttga 12541 ccggtgtgca cggcggtgtg tcctctcggc ttccctcctc cttccctcgc tccccttttt 12601 ctgtgttttt tctttctaac atgattgcgc ctgctctttt tgccccgcag cgtcgcagtg 12661 ggtgtcacct gcgaccgccg tacgttttac tatatacata tttgtttctg tgctgatgcc 12721 atttgtccgc tactccatat cgtgcgtgtt tgttttccag tggtgacgcc ttccgcatca 12781 ttttaatcaa ttcgaggcga aaagggtgat ttgagtgttg gagtgggctt ttttatttat 12841 ttgactccaa acatcttttc ttttcccggt atcttcgggt ccgtcacgca ggtcggtggg 12901 tgtgtccgag tgcgcttctt tactcacgtg ccgctgcgca catacacaga ttttaaaagc 12961 acgcatacac gatctgtgcc ttcaaaacta atcaacaaaa acaaaaatac aaaaaagaca 13021 agaataagag gtcagcaacg cacccacggc tcctttcttc ctgatccacg tcgtggccgc 13081 tgtacgctct caaacacgct ttggcgctga tgcgtgcctt tatacaaaga acaaagaaga 13141 agcgaaaaac gggagcgtgt tgctttgggg atgtatgcgt gccacagttc tgcggtacaa 13201 ttcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca 13261 caacatacga gccggaagca taaagtgtaa atgcatgcgg ccgcacagtg tgtgggtgag 13321 gtgcgcgaag gtgtgtctgt cggtggggga gctcgcgcgg tgtgtgcgtt ggcggctcag 13381 cggcccccct cgcgtctctc acgccggccc gcctgtcccc ctcccccaca taaaaggacg 13441 cagcgcgctc agcttttgtg tgcatgtatg ctatcgctgg tcgcgtgtgc tgcgcctctg 13501 tatcttcttt tgaatctttc cctttttttc gctttctttt atcttcctcc cgtgtaccgc 13561 agttgatgga tcaggcgccc tgttgcaact tgggccgccg acggtgctgg tgcgaggcag 13621 caggccagca tgttgccgag atctttaatt aaaatcccta gcctgcaggg gcgcctgatg 13681 cggtattttc tccttacgca tctgtgcggt atttcacacc gcatacgtca aagcaaccat 13741 agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga 13801 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg 13861 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat 13921 ttagtgcttt acggcacctc gaccccaaaa aacttgattt gggtgatggt tcacgtagtg 13981 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata 14041 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcgggctat tcttttgatt 14101 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat 14161 ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt atggtgcact ctcagtacaa 14221 tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc 14281 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga 14341 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg 14401 tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg 14461 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa 14521 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga 14581 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc 14641 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg 14701 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc 14761 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat 14821 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg 14881 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag 14941 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa 15001 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc 15061 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca 15121 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc 15181 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc 15241 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg 15301 gaagccgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta 15361 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag 15421 gtgcctcact gattaagcat tggtaa // LOCUS pTB008_T7_SSU 11147 bp ds-DNA circular SYN 12-SEP-2016 DEFINITION pTB008_T7_SSU ACCESSION . VERSION . KEYWORDS pT008_T7_SSU SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 11147) AUTHORS . TITLE Direct Submission JOURNAL Exported Friday, Oct 21, 2016 from SnapGene Viewer 3.2.1 http://www.snapgene.com FEATURES Location/Qualifiers source 1..11147 /organism="synthetic DNA construct" /mol_type="other DNA" promoter complement(157..175) /note="T3 promoter" /note="promoter for bacteriophage T3 RNA polymerase" misc_feature 189..787 /note="LPG1 IR" RBS 802..824 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 850..1224 /codon_start=1 /gene="Sh ble from Streptoalloteichus hindustanus" /product="antibiotic-binding protein" /note="BleoR" /note="confers resistance to bleomycin, phleomycin, and Zeocin(TM)" /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR EFALRDPAGNCVHFVAEEQD" misc_feature 1308..1875 /note="1.7K IR" misc_feature 1882..2961 /note="3'SSU" promoter complement(2978..2996) /note="T3 promoter" /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(3017..3033) /note="M13 rev" /note="common sequencing primer, one of multiple similar variants" protein_bind 3041..3057 /bound_moiety="lac repressor encoded by lacI" /note="lac operator" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." promoter complement(3065..3095) /note="lac promoter" /note="promoter for the E. coli lac operon" protein_bind 3110..3131 /bound_moiety="E. coli catabolite activator protein" /note="CAP binding site" /note="CAP binding activates transcription in the presence of cAMP." rep_origin complement(3419..4007) /direction=LEFT /note="ori" /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(4178..5038) /codon_start=1 /gene="bla" /product="beta-lactamase" /note="AmpR" /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" promoter complement(5039..5143) /gene="bla" /note="AmpR promoter" rep_origin 5170..5625 /direction=RIGHT /note="f1 ori" /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind 5766..5782 /note="M13 fwd" /note="common sequencing primer, one of multiple similar variants" promoter 5792..5810 /note="T7 promoter" /note="promoter for bacteriophage T7 RNA polymerase" misc_feature 5824..6702 /note="5'SSU" misc_feature 6709..7097 /note="Calmodulin IR" CDS 7098..9785 /codon_start=1 /note="T7 RNAP" /translation="METINIAKNEFLEPPKKKRKVEFSDIELAAIPFNTLADHYGERLA REQLALEHESYEMGEARFRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEV KAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARF GRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSI HVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQ PCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAW KINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRK DKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTL AKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQ DSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPS ETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAY GVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIW ESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKK PIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHE KYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLD KMPALPAKGNLNLRDILESDFAFA" CDS 7140..7160 /codon_start=1 /product="nuclear localization signal of SV40 large T antigen" /note="SV40 NLS" /translation="PKKKRKV" misc_feature 9789..11126 /note="CYS2-IR" ORIGIN 1 actggctgat ttctacgacc agttcgctga ccagttgcac gagtctcaat tggacaaaat 61 gccagcactt ccggctaaag gtaacttgaa cctccgtgac atcttagagt cggacttcgc 121 gttcgcgtaa cgccaaatca atacgacccg gatctccctt tagtgagggt taattagtcc 181 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg 241 ctactcgggt gtcgcacaca ctgtaaaacg cccccgccgg ctctgtcacg caagaaacga 301 gagcaaaaag accggtagac tatatcacgc acaatcaccg cgtgtgcgtc tccctgggtg 361 aagacaccca tcgcaccctt cgacagccgc ccttatgcct attcaccgtc tgtagaacac 421 acaagaggaa tagcccggtg ccgcgtgcaa gactgcggct tctgcacgca ctatgctcgt 481 ttccgcctct ctctctttgt gcgcgtgtgt gtgtgtgtgt cggagtggcc ctcccgttac 541 gtcttttggg ggtgggtgat agcggcagat gctgcttcga ccttgtgcgc cgcaccggtg 601 ccgttggcta cactgcggaa ggcaacacag aacacaccct gtgccatttc ttcttttttt 661 tttgctttca cccacctttt ccccgtgctt ccccatcttt ccccctcttt ccctaacgta 721 cattgcacct ctccttatcg tgcagtcaca cgctaccact caacgctccc tgcaacactg 781 gagtgagtcg ctagaaataa ttttgtttaa ctttaagaag gagatataca tagtgaccgg 841 atcatcacca tggccaagtt gaccagtgcc gttccggtgc tcaccgcgcg cgacgtcgcc 901 ggagcggtcg agttctggac cgaccggctc gggttctccc gggacttcgt ggaggacgac 961 ttcgccggtg tggtccggga cgacgtgacc ctgttcatca gcgcggtcca ggaccaggtg 1021 gtgccggaca acaccctggc ctgggtgtgg gtgcgcggcc tggacgagct gtacgccgag 1081 tggtcggagg tcgtgtccac gaacttccgg gacgcctccg ggccggccat gaccgagatc 1141 ggcgagcagc cgtgggggcg ggagttcgcc ctgcgcgacc cggccggcaa ctgcgtgcac 1201 ttcgtggccg aggagcagga ctgaccgacg ccgaccaaca ccgccggtcc gacggcggcc 1261 cacgggtccc aggctagttc tagttctagg gggcgcgaat tcagatcctc gtgtgagcgt 1321 tcgcggaatc ggtcgctcgt gtttatgccc gtcttggtgt tgtgctcgca aggcggtgca 1381 gcaggatacc gtcgccctcc tctctccttg cttctctgtt cttcaattcg cgatctcaca 1441 gaggccggct gtgcacgccc ttcctcaccc tccttttccc acctctcggc caccggtcgg 1501 ctccgttccg tctgccgtcg agaagggacg ggcatgtgca gctcctccct ttctctcgcg 1561 cgcgcatctt ctcttgcttg tggcactcac gctcatgcgt caaggcggcc ccacgcgagc 1621 ccctgcgctc ccttccctct tgcgcatccg tagccggact ggtcgatgcg caaggccggc 1681 atgaaggagc gcgtgccctc aagagcggac tatcatgccc tacgtgggcc acgcagcgat 1741 gaggccggct tcgcggagat gcgtcacgca cgtgccagat gatgccgtac gccttccttg 1801 acttgcgccc ccctctcttc ctccgtctct cactctctct ctctcacaca cacacacaca 1861 cacacacaca cacaaagctc cggttcgtcc ggccgtaacg ccttttcaac tcacggcctc 1921 taggaatgaa ggagggtagt tcgggggaga acgtactggg gcgtcagagg tgaaattctt 1981 agaccgcacc aagacgaact acagcgaagg cattcttcaa ggataccttc ctcaatcaag 2041 aaccaaagtg tggagatcga agatgattag agaccattgt agtccacact gcaaacgatg 2101 acacccatga attggggatc ttatgggccg gcctgcggca gggtttaccc tgtgtcagca 2161 ccgcgcccgc ttttaccaac ttacgtatct tttctattcg gcctttaccg gccacccacg 2221 ggaatatcct cagcacgttt tctgtttttt cacgcgaaag ctttgaggtt acagtctcag 2281 gggggagtac gttcgcaaga gtgaaactta aagaaattga cggaatggca ccacaagacg 2341 tggagcgtgc ggtttaattt gactcaacac ggggaacttt accagatccg gacaggatga 2401 ggattgacag attgagtgtt ctttctcgat tccctgaatg gtggtgcatg gccgcttttg 2461 gtcggtggag tgatttgttt ggttgattcc gtcaacggac gagatccaag ctgcccagta 2521 gaattcagaa ttgcccatag aatagcaaac tcatcggcgg gttttaccca acggtgggcc 2581 gcattcggtc gaattcttct ctgcgggatt cctttgtaat tgcacaaggt gaaattttgg 2641 gcaacagcag gtctgtgatg ctcctcaatg ttctgggcga cacgcgcact acaatgtcag 2701 tgagaacaag aaaaacgact tttgtcgaac ctacttgatc aaaagagtgg ggaaaccccg 2761 gaatcacata gacccacttg ggaccgagga ttgcaattat tggtcgcgca acgaggaatg 2821 tctcgtaggc gcagctcatc aaactgtgcc gattacgtcc ctgccatttg tacacaccgc 2881 ccgtcgttgt ttccgatgat ggtgcaatac aggtgatcgg acaggcggtg ttttatccgc 2941 ccgaaagttc accgatattt aaatccagct tttgttccct ttagtgaggg ttaattgcgc 3001 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc 3061 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct 3121 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc 3181 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt 3241 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag 3301 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca 3361 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt 3421 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc 3481 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct 3541 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg 3601 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca 3661 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact 3721 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta 3781 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta 3841 actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct 3901 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt 3961 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga 4021 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca 4081 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat 4141 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg 4201 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt 4261 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag 4321 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc 4381 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag 4441 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca 4501 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa 4561 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga 4621 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata 4681 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca 4741 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg 4801 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg 4861 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg 4921 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag 4981 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac 5041 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca 5101 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag 5161 tgccacctga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca 5221 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct 5281 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt 5341 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac 5401 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct 5461 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt 5521 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac 5581 aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttccatt cgccattcag 5641 gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagctggc 5701 gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt cccagtcacg 5761 acgttgtaaa acgacggcca gtgagcgcgc gtaatacgac tcactatagg gcgaattgga 5821 tttaaattgg ataacttggc gaaacgccaa gctaatacat gaaccaaccg ggtgttctcc 5881 actccagacg gtgggcaacc atcgtcgtga gacgcccagc gaatgaatga cagtaaaacc 5941 aatgccttca ctggcagtaa cacccagcag tgttgactca attcattccg tgcgaaagcc 6001 ggcttgttcc ggcgtctttt gacgaacaac tgccctatca gctggtgatg gccgtgtagt 6061 ggactgccat ggcgttgacg ggagcggggg attagggttc gattccggag agggagcctg 6121 agaaatagct accacttcta cggagggcag caggcgcgca aattgcccaa tgtcaaaaca 6181 aaacgatgag gcagcgaaaa gaaatagagt tgtcagtcca tttggattgt catttcaatg 6241 ggggatattt aaacccatcc aatatcgagt aacaattgga ggacaagtct ggtgccagca 6301 cccgcggtaa ttccagctcc aaaagcgtat attaatgctg ttgctgttaa agggttcgta 6361 gttgaactgt gggctgtgca ggtttgttcc tggtcgtccc gtccatgtcg gatttggtga 6421 cccaggccct tgcagcccgt gaacattcaa agaaacaaga aacacgggag tggttccttt 6481 cctgatttac gcatgtcatg catgccaggg ggcgtccgtg attttttact gtgactaaag 6541 aagcgtgact aaagcagtca tttgacttga attagaaagc atgggataac aaaggagcag 6601 cctctaggct accgtttcgg cttttgttgg ttttaaaggt ctattggaga ttatggagct 6661 gtgcgacaag tgctttccca tcgcaacttc ggttcggtgt gtggcgcccg acacacgaag 6721 cgtgtgatga atgaaaagga ggagacactc gacacttgaa tatttctccc tatcaagccc 6781 tttcaggcct gcgtgctccc tcaacccctt tctcgataac gtgcgacgta atgttacatg 6841 cacacacgtc gcacatctcc tgtgaatggc actgccttct cgtgccgtca ccatgcccgc 6901 ctttcagcac tggctcttcg tttcttcacc gcttcttcgt tctgtgaacc cgcgcgcacg 6961 cacgcatcga agggattcat cgccattgcc ctctccgcct ctctcgctcg ctgcacattg 7021 actctccaca gccccccctc cctcgcacag ccagccccac ccacccaccc accgctctac 7081 acacctccac agcagccatg gagacgatta acatcgctaa gaacgaattc ctcgagcctc 7141 caaaaaagaa gagaaaggtc gaattctctg acatcgaact ggctgctatc ccgttcaaca 7201 ctctggctga ccattacggt gagcgtttag ctcgcgaaca gttggccctt gagcatgagt 7261 cttacgagat gggtgaagca cgcttccgca agatgtttga gcgtcaactt aaagctggtg 7321 aggttgcgga taacgctgcc gccaagcctc tcatcactac cctactccct aagatgattg 7381 cacgcatcaa cgactggttt gaggaagtga aagctaagcg cggcaagcgc ccgacagcct 7441 tccagttcct gcaagaaatc aagccggaag ccgtagcgta catcaccatt aagaccactc 7501 tggcttgcct aaccagtgct gacaatacaa ccgttcaggc tgtagcaagc gcaatcggtc 7561 gggccattga ggacgaggct cgcttcggtc gtatccgtga ccttgaagct aagcacttca 7621 agaaaaacgt tgaggaacaa ctcaacaagc gcgtagggca cgtctacaag aaagcattta 7681 tgcaagttgt cgaggctgac atgctctcta agggtctact cggtggcgag gcgtggtctt 7741 cgtggcataa ggaagactct attcatgtag gagtacgctg catcgagatg ctcattgagt 7801 caaccggaat ggttagctta caccgccaaa atgctggcgt agtaggtcaa gactctgaga 7861 ctatcgaact cgcacctgaa tacgctgagg ctatcgcaac ccgtgcaggt gcgctggctg 7921 gcatctctcc gatgttccaa ccttgcgtag ttcctcctaa gccgtggact ggcattactg 7981 gtggtggcta ttgggctaac ggtcgtcgtc ctctggcgct ggtgcgtact cacagtaaga 8041 aagcactgat gcgctacgaa gacgtttaca tgcctgaggt gtacaaagcg attaacattg 8101 cgcaaaacac cgcatggaaa atcaacaaga aagtcctagc ggtcgccaac gtaatcacca 8161 agtggaagca ttgtccggtc gaggacatcc ctgcgattga gcgtgaagaa ctcccgatga 8221 aaccggaaga catcgacatg aatcctgagg ctctcaccgc gtggaaacgt gctgccgctg 8281 ctgtgtaccg caaggacaag gctcgcaagt ctcgccgtat cagccttgag ttcatgcttg 8341 agcaagccaa taagtttgct aaccataagg ccatctggtt cccttacaac atggactggc 8401 gcggtcgtgt ttacgctgtg tcaatgttca acccgcaagg taacgatatg accaaaggac 8461 tgcttacgct ggcgaaaggt aaaccaatcg gtaaggaagg ttactactgg ctgaaaatcc 8521 acggtgcaaa ctgtgcgggt gtcgataagg ttccgttccc tgagcgcatc aagttcattg 8581 aggaaaacca cgagaacatc atggcttgcg ctaagtctcc actggagaac acttggtggg 8641 ctgagcaaga ttctccgttc tgcttccttg cgttctgctt tgagtacgct ggggtacagc 8701 accacggcct gagctataac tgctcccttc cgctggcgtt tgacgggtct tgctctggca 8761 tccagcactt ctccgcgatg ctccgagatg aggtaggtgg tcgcgcggtt aacttgcttc 8821 ctagtgaaac cgttcaggac atctacggga ttgttgctaa gaaagtcaac gagattctac 8881 aagcagacgc aatcaatggg accgataacg aagtagttac cgtgaccgat gagaacactg 8941 gtgaaatctc tgagaaagtc aagctgggca ctaaggcact ggctggtcaa tggctggctt 9001 acggtgttac tcgcagtgtg actaagcgtt cagtcatgac gctggcttac gggtccaaag 9061 agttcggctt ccgtcaacaa gtgctggaag ataccattca gccagctatt gattccggca 9121 agggtctgat gttcactcag ccgaatcagg ctgctggata catggctaag ctgatttggg 9181 aatctgtgag cgtgacggtg gtagctgcgg ttgaagcaat gaactggctt aagtctgctg 9241 ctaagctgct ggctgctgag gtcaaagata agaagactgg agagattctt cgcaagcgtt 9301 gcgctgtgca ttgggtaact cctgatggtt tccctgtgtg gcaggaatac aagaagccta 9361 ttcagacgcg cttgaacctg atgttcctcg gtcagttccg cttacagcct accattaaca 9421 ccaacaaaga tagcgagatt gatgcacaca aacaggagtc tggtatcgct cctaactttg 9481 tacacagcca agacggtagc caccttcgta agactgtagt gtgggcacac gagaagtacg 9541 gaatcgaatc ttttgcactg attcacgact ccttcggtac cattccggct gacgctgcga 9601 acctgttcaa agcagtgcgc gaaactatgg ttgacacata tgagtcttgt gatgtactgg 9661 ctgatttcta cgaccagttc gctgaccagt tgcacgagtc tcaattggac aaaatgccag 9721 cacttccggc taaaggtaac ttgaacctcc gtgacatctt agagtcggac ttcgcgttcg 9781 cgtagtctag aggatcggag gtgtgtgtgc ccttgtgtgc tgtgtgtggg tggacgcagc 9841 gatgcccggc gcgtgtgggc acctccttgg gtgcgcgccc gccgtggcag ctgcgcgtgc 9901 gtgcgagatg tgaggcagag gaagaggaag gcgatgcggg cgacacgcag aggtgcggcg 9961 gacgtagggg ggaaatggac gagcaggcgc gctgtgaatc ggagctgcgg caccacccaa 10021 gtcgtggtgc cccgcgaatg gctgttctgc cgccctcgct tcacgcctcc ccctcccctc 10081 gcgtgccctc gcgtggcctc ccttgttatc cctctctctc gcacgcacac ggatacgcga 10141 gcccgctatt ctgccttcgt ctggctcttt gtattctgct tgcttcttca gcacacttgt 10201 gtgctgtgcg ttcagcgata tcttccacta ctttgttttc tcctccccct cgggaggtgc 10261 ttcgcttgtg ctttgacggt ggtgcgtggc tgctgggtca tgtgccgggc gtgcgcgcct 10321 ccgccgcctc cctgcagctt gtgggtctgg ctgcgttcgc accgcgctcg cgtgcatgca 10381 catgcctgca ctgcgtcggg aacgacttcc gggcgcgttg gccccccgcc tctgcagcca 10441 cggtctgttt attgattgtg cttgcttcat cggctcttct ctgcgcgcgt gcgtgcgtgc 10501 gtgtgcgtgt ccgtgcgtat gcgtgaggcg caacggtccc cagagcaagg catgtcgagg 10561 ggaacactat agacgcatgt gtacgtgtac acgatgtgta tacgtatacg tgtaccgaat 10621 ggtgcgtgcg cgtgtgcagc attgccgtga cggcatgtac gaagcgctgc agtgggatgg 10681 accctgtgcg cgtgccggag aggtagtgtc gcgtgtgggt gcggagtgat ggaggctagg 10741 gggcttacga gcaccgtcgc ttttcccccg atggcggctg gcacgcagcg cacgcaccgg 10801 ggatgtgtga cgtgcgtcct gtgcgcctct ccctctcccc ttgtcgccgg cgcatggatg 10861 caccgctgtt gtgtgaggtt gcccgcacct gcgttgttgc ctgtgatgac gtccctccct 10921 ctcttgcact ctccccgtcc ccacctgccc tgcaccgtgg tcgactgctc ccgacgccct 10981 gcacagactc tcgtcgccac caccagcagc agccctctat atacccgcca ctgccgtagc 11041 gttcgggccg tggctctgcg tttcacttgc tctcccctcg ctctgttcat tgcttccttc 11101 tgttcccctc gctgcccgcg tccggagatc tatgagtctt gtgatgt // LOCUS pTB011_Cas9_Tbrucei_Tub 10042 bp ds-DNA circular SYN 21-OCT-2016 DEFINITION pTB011_Cas9_Tbrucei_Tub ACCESSION . VERSION . KEYWORDS pTB011_Cas9_Tbrucei_Tub SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 10042) AUTHORS . TITLE Direct Submission JOURNAL Exported Friday, Oct 21, 2016 from SnapGene Viewer 3.2.1 http://www.snapgene.com FEATURES Location/Qualifiers source 1..10042 /organism="synthetic DNA construct" /mol_type="other DNA" 3'UTR 11..388 /note="Alpha tub" CDS 395..994 /codon_start=1 /note="PuroR" /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA" misc_feature 1001..1149 /note="Ald 3'UTR" misc_feature 1156..1344 /note="Aldolase 5'" misc_feature 1355..1384 /note="GS linker" CDS 1394..1459 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /note="3xFLAG" /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 1466..1486 /codon_start=1 /product="nuclear localization signal of SV40 large T antigen" /note="SV40 NLS" /translation="PKKKRKV" CDS 1511..5611 /codon_start=1 /product="Cas9 (Csn1) endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /note="Cas9" /note="generates RNA-guided double strand breaks in DNA" /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGD" CDS 5612..5659 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /note="nucleoplasmin NLS" /translation="KRPAATKKAGQAKKKK" misc_feature 5708..6058 /note="PFR2 IGS" misc_feature 6065..6192 /note="Actin 5'" CDS 6199..6588 /codon_start=1 /note="BlastR" /translation="MPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTGVN VYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGIKA IVKDSDGQPTAVGIRELLPSGYVWEG" 5'UTR 6592..7141 /note="Beta Tub" promoter complement(7167..7185) /note="SP6 promoter" /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind complement(7203..7219) /note="M13 rev" /note="common sequencing primer, one of multiple similar variants" protein_bind 7227..7243 /bound_moiety="lac repressor encoded by lacI" /note="lac operator" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." promoter complement(7251..7281) /note="lac promoter" /note="promoter for the E. coli lac operon" protein_bind 7296..7317 /bound_moiety="E. coli catabolite activator protein" /note="CAP binding site" /note="CAP binding activates transcription in the presence of cAMP." rep_origin complement(7605..8193) /direction=LEFT /note="ori" /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(8364..9224) /codon_start=1 /gene="bla" /product="beta-lactamase" /note="AmpR" /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" promoter complement(9225..9329) /gene="bla" /note="AmpR promoter" rep_origin complement(9407..9862) /direction=LEFT /note="f1 ori" /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind 10003..10019 /note="M13 fwd" /note="common sequencing primer, one of multiple similar variants" promoter 10026..2 /note="T7 promoter" /note="promoter for bacteriophage T7 RNA polymerase" ORIGIN 1 gggcgaattg ggcccgacgt cgcatgctcc cggccgccat ggccgcggga ttttaattaa 61 tagaaagtgt gacaacgtcg caccatgtgt aggttttcat ttatgttctt tctttctttt 121 ttttgtgaat ttgttttctg tctcaaatgt ttttaattcg cttgggacct atgtttttct 181 tgtttttttg ctcacccttt gtgtaggagg caccctgtca cgtctgtggt tgcgtgtatg 241 ccttccttcc ccttattcgc ttcttcctgt cgtgtcacac ctctttctct ctctcccttt 301 ccgccttttc tttcaatctt gttttctcga ccagccctac tagaggagaa agaatagtaa 361 ccctttcatc aaagaaaata gttcaaacga attcatgact gaatacaagc caacggttcg 421 tcttgctacg cgagacgatg tgccgcgggc cgtacggacg ctggctgctg cgttcgctga 481 ttatcccgcc acccgacata cggttgaccc agatcgacac attgagcgag tcacagaact 541 tcaagagtta ttcttaacac gggtgggctt ggacattggt aaggtttggg ttgcggacga 601 tggcgcggct gttgcggtgt ggacgacgcc agagtcagtc gaagcgggcg cagtgtttgc 661 tgagatcggt ccccgaatgg cggagcttag cgggtcacgc ctcgctgcac aacagcagat 721 ggaaggtctg ttggcaccac atagacctaa ggagccggct tggttcttag cgacagttgg 781 cgtcagtccg gaccaccaag gtaaggggct gggttcagcg gttgttcttc caggagtaga 841 ggctgcggaa agggcaggtg tgccggcgtt ccttgaaacg tctgcaccac gtaaccttcc 901 tttttacgag agattggggt ttacagtaac agcagatgtt gaagtgcctg aagggccgag 961 gacatggtgc atgacacgta agccgggagc ctaaccatgg tgcccattta gttggctttt 1021 cccttgtctc gtgtcttttc cgtggaaagg ttcccggagt aatctgatgg cacagcaggg 1081 aggtgcgcct gcaggttggt taggaagggg ggatgatgta aaagaagaaa atggggggat 1141 tcgagcccca cgcgttgctc aagctgtgta gcgcaggcgt ttccttacat atttctctaa 1201 caggcacgga agcctaacaa atacacttgg cttatttttt tgccccctca tgtcttgtac 1261 aaatatttgc gatagcttag ctatcagcca cattaatcaa acaagtatac caacaagccc 1321 gaaaacagaa actcaactgc aacgggtacc tatgggttct ggtagtggtt ccggttccgg 1381 ttctaagctt atggactata aggaccacga cggagactac aaggatcatg atattgatta 1441 caaagacgat gacgataaga tggccccaaa gaagaagcgg aaggtcggta tccacggagt 1501 cccagcagcc gacaagaagt acagcatcgg cctggacatc ggcaccaact ctgtgggctg 1561 ggccgtgatc accgacgagt acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac 1621 cgaccggcac agcatcaaga agaacctgat cggagccctg ctgttcgaca gcggcgaaac 1681 agccgaggcc acccggctga agagaaccgc cagaagaaga tacaccagac ggaagaaccg 1741 gatctgctat ctgcaagaga tcttcagcaa cgagatggcc aaggtggacg acagcttctt 1801 ccacagactg gaagagtcct tcctggtgga agaggataag aagcacgagc ggcaccccat 1861 cttcggcaac atcgtggacg aggtggccta ccacgagaag taccccacca tctaccacct 1921 gagaaagaaa ctggtggaca gcaccgacaa ggccgacctg cggctgatct atctggccct 1981 ggcccacatg atcaagttcc ggggccactt cctgatcgag ggcgacctga accccgacaa 2041 cagcgacgtg gacaagctgt tcatccagct ggtgcagacc tacaaccagc tgttcgagga 2101 aaaccccatc aacgccagcg gcgtggacgc caaggccatc ctgtctgcca gactgagcaa 2161 gagcagacgg ctggaaaatc tgatcgccca gctgcccggc gagaagaaga atggcctgtt 2221 cggaaacctg attgccctga gcctgggcct gacccccaac ttcaagagca acttcgacct 2281 ggccgaggat gccaaactgc agctgagcaa ggacacctac gacgacgacc tggacaacct 2341 gctggcccag atcggcgacc agtacgccga cctgtttctg gccgccaaga acctgtccga 2401 cgccatcctg ctgagcgaca tcctgagagt gaacaccgag atcaccaagg cccccctgag 2461 cgcctctatg atcaagagat acgacgagca ccaccaggac ctgaccctgc tgaaagctct 2521 cgtgcggcag cagctgcctg agaagtacaa agagattttc ttcgaccaga gcaagaacgg 2581 ctacgccggc tacattgacg gcggagccag ccaggaagag ttctacaagt tcatcaagcc 2641 catcctggaa aagatggacg gcaccgagga actgctcgtg aagctgaaca gagaggacct 2701 gctgcggaag cagcggacct tcgacaacgg cagcatcccc caccagatcc acctgggaga 2761 gctgcacgcc attctgcggc ggcaggaaga tttttaccca ttcctgaagg acaaccggga 2821 aaagatcgag aagatcctga ccttccgcat cccctactac gtgggccctc tggccagggg 2881 aaacagcaga ttcgcctgga tgaccagaaa gagcgaggaa accatcaccc cctggaactt 2941 cgaggaagtg gtggacaagg gcgcttccgc ccagagcttc atcgagcgga tgaccaactt 3001 cgataagaac ctgcccaacg agaaggtgct gcccaagcac agcctgctgt acgagtactt 3061 caccgtgtat aacgagctga ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc 3121 cttcctgagc ggcgagcaga aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa 3181 agtgaccgtg aagcagctga aagaggacta cttcaagaaa atcgagtgct tcgactccgt 3241 ggaaatctcc ggcgtggaag atcggttcaa cgcctccctg ggcacatacc acgatctgct 3301 gaaaattatc aaggacaagg acttcctgga caatgaggaa aacgaggaca ttctggaaga 3361 tatcgtgctg accctgacac tgtttgagga cagagagatg atcgaggaac ggctgaaaac 3421 ctatgcccac ctgttcgacg acaaagtgat gaagcagctg aagcggcgga gatacaccgg 3481 ctggggcagg ctgagccgga agctgatcaa cggcatccgg gacaagcagt ccggcaagac 3541 aatcctggat ttcctgaagt ccgacggctt cgccaacaga aacttcatgc agctgatcca 3601 cgacgacagc ctgaccttta aagaggacat ccagaaagcc caggtgtccg gccagggcga 3661 tagcctgcac gagcacattg ccaatctggc cggcagcccc gccattaaga agggcatcct 3721 gcagacagtg aaggtggtgg acgagctcgt gaaagtgatg ggccggcaca agcccgagaa 3781 catcgtgatc gaaatggcca gagagaacca gaccacccag aagggacaga agaacagccg 3841 cgagagaatg aagcggatcg aagagggcat caaagagctg ggcagccaga tcctgaaaga 3901 acaccccgtg gaaaacaccc agctgcagaa cgagaagctg tacctgtact acctgcagaa 3961 tgggcgggat atgtacgtgg accaggaact ggacatcaac cggctgtccg actacgatgt 4021 ggaccatatc gtgcctcaga gctttctgaa ggacgactcc atcgacaaca aggtgctgac 4081 cagaagcgac aagaaccggg gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa 4141 gatgaagaac tactggcggc agctgctgaa cgccaagctg attacccaga gaaagttcga 4201 caatctgacc aaggccgaga gaggcggcct gagcgaactg gataaggccg gcttcatcaa 4261 gagacagctg gtggaaaccc ggcagatcac aaagcacgtg gcacagatcc tggactcccg 4321 gatgaacact aagtacgacg agaatgacaa gctgatccgg gaagtgaaag tgatcaccct 4381 gaagtccaag ctggtgtccg atttccggaa ggatttccag ttttacaaag tgcgcgagat 4441 caacaactac caccacgccc acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat 4501 caaaaagtac cctaagctgg aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt 4561 gcggaagatg atcgccaaga gcgagcagga aatcggcaag gctaccgcca agtacttctt 4621 ctacagcaac atcatgaact ttttcaagac cgagattacc ctggccaacg gcgagatccg 4681 gaagcggcct ctgatcgaga caaacggcga aaccggggag atcgtgtggg ataagggccg 4741 ggattttgcc accgtgcgga aagtgctgag catgccccaa gtgaatatcg tgaaaaagac 4801 cgaggtgcag acaggcggct tcagcaaaga gtctatcctg cccaagagga acagcgataa 4861 gctgatcgcc agaaagaagg actgggaccc taagaagtac ggcggcttcg acagccccac 4921 cgtggcctat tctgtgctgg tggtggccaa agtggaaaag ggcaagtcca agaaactgaa 4981 gagtgtgaaa gagctgctgg ggatcaccat catggaaaga agcagcttcg agaagaatcc 5041 catcgacttt ctggaagcca agggctacaa agaagtgaaa aaggacctga tcatcaagct 5101 gcctaagtac tccctgttcg agctggaaaa cggccggaag agaatgctgg cctctgccgg 5161 cgaactgcag aagggaaacg aactggccct gccctccaaa tatgtgaact tcctgtacct 5221 ggccagccac tatgagaagc tgaagggctc ccccgaggat aatgagcaga aacagctgtt 5281 tgtggaacag cacaagcact acctggacga gatcatcgag cagatcagcg agttctccaa 5341 gagagtgatc ctggccgacg ctaatctgga caaagtgctg tccgcctaca acaagcaccg 5401 ggataagccc atcagagagc aggccgagaa tatcatccac ctgtttaccc tgaccaatct 5461 gggagcccct gccgccttca agtactttga caccaccatc gaccggaaga ggtacaccag 5521 caccaaagag gtgctggacg ccaccctgat ccaccagagc atcaccggcc tgtacgagac 5581 acggatcgac ctgtctcagc tgggaggcga caaaaggccg gcggccacga aaaaggccgg 5641 ccaggcaaaa aagaaaaagt aaggatccgg atcaggatct ggatcaggat cgggtagtta 5701 agagctccgc tgcgcttaaa tgtctttcat tataatcaat gtataacctt tatgtagtat 5761 ttcaatctat gccgctgtgt acgtgcactg cggtgcctat ccttcggcat tagagagtca 5821 ctgtttgtgt agatcgtagc tgcatgtctg acatctggtt cattagtgcc gttttgagtc 5881 tgcactgttg tgcaacttga tattcatcac tgcacaccaa ccgtgtgttt cctcctttgg 5941 gtgcaccctt taatccttgt cttctccttt tttgtctctt tccccctcga aaaggtgtca 6001 aactactgcc gcataaacta cggttatccc aaatttaaga gaaagcaata aagcatcagc 6061 atgcaggcta gggcacagca aggtcttctg aaattcatgt tttttttttt tttactctgc 6121 attgcagtct ccgctcttat ttagttttgc tttacgtaag gtctcgttgc tgccataaaa 6181 taagctctag tggctagtat gcctttgtct caagaagaat ccaccctcat tgaaagagca 6241 acggctacaa tcaacagcat ccccatctct gaagactaca gcgtcgccag cgcagctctc 6301 tctagcgacg gccgcatctt cactggtgtc aatgtatatc attttactgg gggaccttgt 6361 gcagaactcg tggtgctggg cactgctgct gctgcggcag ctggcaacct gacttgtatc 6421 gtcgcgatcg gaaatgagaa caggggcatc ttgagcccct gcggacggtg ccgacaggtg 6481 cttctcgatc tgcatcctgg gatcaaggcc atagtgaagg acagtgatgg acagccgacg 6541 gcagttggga ttcgtgaatt gctgccctct ggttatgtgt gggagggcta attcgaatag 6601 acgcggacgg ggcatttccc gttcgtcatt agcagtaggt aatgaagatg tttgtttctc 6661 gtcccctttc tccttcgtcc ttctgtcatt ttgttctttt gtgtttatgt tttgttgttg 6721 ttttctttaa tttttttttt cttccacgtt tgtgtacatc cgcgcgccac tctattcaga 6781 gagccacgga tagtagagga ggtgggaagg gtatatgagg gacacgcgta ccatgatgtg 6841 ggatgtattg gggtccctgt ctgtccttac gtgactatgt atgaaccgtc acgtgtaaga 6901 tgagctagtg agatcaacag tacaactcat caacacgcct tcttctcgtt aaatgtacac 6961 aatcttgatc ctccaccttt atgggtccca ttgtttgcct cttccgctgt gtggagtgcg 7021 cctacacgca cttctcactt cgtaagtggt ggtggcgtaa gtattgccta atgttgactc 7081 tatattctcc tctcctcacc ccctcgcggt gctgatttct gacttaatta aaatcactag 7141 tgcggccgcc tgcatagctt gagtattcta tagtgtcacc taaatagctt ggcgtaatca 7201 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga 7261 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt 7321 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga 7381 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc 7441 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg 7501 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc 7561 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc 7621 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga 7681 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc 7741 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat 7801 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg 7861 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc 7921 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga 7981 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact 8041 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt 8101 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag 8161 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg 8221 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa 8281 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata 8341 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg 8401 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata 8461 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg 8521 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct 8581 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt 8641 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc 8701 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga 8761 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt 8821 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc 8881 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa 8941 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca 9001 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca 9061 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct 9121 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc 9181 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa 9241 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt 9301 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgatgcg 9361 gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggaaat tgtaagcgtt 9421 aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag 9481 gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt 9541 gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga 9601 aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg 9661 gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct 9721 tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc 9781 gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt 9841 aatgcgccgc tacagggcgc gtccattcgc cattcaggct gcgcaactgt tgggaagggc 9901 gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc 9961 gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg 10021 aattgtaata cgactcacta ta // LOCUS pTBlast 4595 bp ds-DNA circular SYN 06-JUN-2016 DEFINITION pTBlast ACCESSION . VERSION . KEYWORDS pTBlast SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4595) AUTHORS . TITLE Direct Submission JOURNAL Exported Tuesday, Jun 7, 2016 from SnapGene Viewer 3.1.2 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4595 /organism="synthetic DNA construct" /mol_type="other DNA" promoter complement(14..32) /note="T3 promoter" /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(53..69) /note="M13 rev" /note="common sequencing primer, one of multiple similar variants" protein_bind 77..93 /bound_moiety="lac repressor encoded by lacI" /note="lac operator" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." promoter complement(101..131) /note="lac promoter" /note="promoter for the E. coli lac operon" protein_bind 146..167 /bound_moiety="E. coli catabolite activator protein" /note="CAP binding site" /note="CAP binding activates transcription in the presence of cAMP." rep_origin complement(455..1043) /direction=LEFT /note="ori" /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(1214..2074) /codon_start=1 /gene="bla" /product="beta-lactamase" /note="AmpR" /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" promoter complement(2075..2179) /gene="bla" /note="AmpR promoter" rep_origin 2206..2662 /direction=RIGHT /note="f1 ori" /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind 2803..2819 /note="M13 fwd" /note="common sequencing primer, one of multiple similar variants" promoter 2829..2847 /note="T7 promoter" /note="promoter for bacteriophage T7 RNA polymerase" misc_feature 2865..2884 /note="#161 (N-terminal For)" misc_feature 2885..3321 /note="5'60S acidic ribosomal protein P2 (LmxM.15)" CDS 3322..3711 /codon_start=1 /note="BlastR" /translation="MPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTGVN VYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGIKA IVKDSDGQPTAVGIRELLPSGYVWEG" misc_feature complement(3345..3367) /note="BlastR_518A" misc_feature complement(3618..3639) /note="reverse 518" misc_feature 3712..4567 /note="3'glutamine synthetase (LmxM.06)" misc_feature complement(4568..4587) /note="#162 (C-terminal Rev)" ORIGIN 1 ccagcttttg ttccctttag tgagggttaa ttgcgcgctt ggcgtaatca tggtcatagc 61 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca 121 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct 181 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac 241 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc 301 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt 361 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg 421 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg 481 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat 541 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta 601 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct 661 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc 721 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa 781 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg 841 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag 901 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt 961 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta 1021 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc 1081 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca 1141 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa 1201 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat 1261 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct 1321 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt 1381 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat 1441 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta 1501 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg 1561 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt 1621 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg 1681 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg 1741 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc 1801 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa 1861 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac 1921 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt 1981 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg 2041 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa 2101 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata 2161 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgcg ccctgtagcg 2221 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg 2281 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc 2341 cccgtcaagc tctaaatcgg gggcgccctt tagggttccg atttagtgct ttacggcacc 2401 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga 2461 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa 2521 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga 2581 tttcggccta ttggttaaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac 2641 aaaatattaa cgcttacaat ttccattcgc cattcaggct gcgcaactgt tgggaagggc 2701 gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc 2761 gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg 2821 agcgcgcgta atacgactca ctatagggcg aattggattt aaatgtataa tgcagacctg 2881 ctgccggatg aaaacgagag agagacttga gcggtctccg cgagcggagc aggggacggg 2941 gggagtgatg atggcgagca gtcaaggcat gcctgtctct ctgtctctct ttggccgtgt 3001 ggcaccccgg tggaggcatc accctcccca ccctggcccc gccctcccca catggatggt 3061 ccgccatgtg tgcacagcct gtggttgtat gcctttatac gaacctttgc tactgttcct 3121 cgcacgccca cgactttacc gaacgtctct cccccctccc gcacgcatcc gtgccaaccg 3181 cacaacgcgc gcgcgcgcac gcacgcaacc cacacacata tatctgcatc tatacgccac 3241 accacccacc gctactcggg tgtcgacgtc ggcgaaagct ttccgtttcg ttctacgcag 3301 tcctcgtatc ctcgctccac catgcctttg tctcaagaag aatccaccct cattgaaaga 3361 gcaacggcta caatcaacag catccccatc tctgaagact acagcgtcgc cagcgcagct 3421 ctctctagcg acggccgcat cttcactggt gtcaatgtat atcattttac tgggggacct 3481 tgtgcagaac tcgtggtgct gggcactgct gctgctgcgg cagctggcaa cctgacttgt 3541 atcgtcgcga tcggaaatga gaacaggggc atcttgagcc cctgcggacg gtgtcgacag 3601 gtgcttctcg atctgcatcc tgggatcaaa gcgatagtga aggacagtga tggacagccg 3661 acggcagttg ggattcgtga attgctgccc tctggttatg tgtgggaggg catagaagcc 3721 acaccgcgtg agcttgcgtc agtatctcgc agtccccgag ttgtgaactc tttgactgac 3781 tcatccgtat gacgaaagtt ctattgcaaa ttcgtttggg gtatttgaat tggatacctt 3841 gcctatttca aaattatatc tacaacatca gtaccggtct tctgcggagt tagagggcac 3901 aaagaaacac tggcgatcca aaaaaaaatt gtaaaagata aacggtaacg gataaatgat 3961 tctcttcctg tacgcctatt gctgtgactc tttatggaga cacaagcgtg ttcagccagt 4021 gcgagaattg tttgtgccgt tataattaaa aaaaaagaaa caatgaggtc gcatctcttc 4081 catttaaaag ttttttttca ttcattcttt gaccaaaaga acctattttt cgctatggcc 4141 caacttggtt gaaccgtttg gcatagagat ggactttctc ttgagtgttt aaaaggagtg 4201 aggtctgtga aaagtaacac gaccgtgaat gctgtagcat atttgctttt ttttcgtttc 4261 ccaaggagga tttccatcga tagggtctct tttcggtacc gtcgagaatg tttggggtcg 4321 aatcgccaat tgcgagaaac tgacaggcag tgggtctgaa tgctgaagta ttgcaagcta 4381 acggcatcct ttgctcaggc atactttttt tttacgatgt cccatacctt gatgaggact 4441 aaggcagtgg gtgtttccca ctccaatgag tttggggttt gtactgcaat gttgttggca 4501 tttgatctct tagctaccac tgcgtctttt cctggccttc ttgttctctc caactgacac 4561 actggttgca caggtctctc aaattggatt taaat // LOCUS pTNeo 4629 bp ds-DNA circular SYN 16-MAY-2016 DEFINITION pTNeo ACCESSION . VERSION . KEYWORDS pTNeo SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4629) AUTHORS . TITLE Direct Submission JOURNAL Exported Tuesday, Jun 7, 2016 from SnapGene Viewer 3.1.2 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4629 /organism="synthetic DNA construct" /mol_type="other DNA" promoter complement(14..32) /note="T3 promoter" /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(53..69) /note="M13 rev" /note="common sequencing primer, one of multiple similar variants" protein_bind 77..93 /bound_moiety="lac repressor encoded by lacI" /note="lac operator" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." promoter complement(101..131) /note="lac promoter" /note="promoter for the E. coli lac operon" protein_bind 146..167 /bound_moiety="E. coli catabolite activator protein" /note="CAP binding site" /note="CAP binding activates transcription in the presence of cAMP." rep_origin complement(455..1043) /direction=LEFT /note="ori" /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(1214..2074) /codon_start=1 /gene="bla" /product="beta-lactamase" /note="AmpR" /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" promoter complement(2075..2179) /gene="bla" /note="AmpR promoter" rep_origin 2206..2662 /direction=RIGHT /note="f1 ori" /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind 2803..2819 /note="M13 fwd" /note="common sequencing primer, one of multiple similar variants" promoter 2829..2847 /note="T7 promoter" /note="promoter for bacteriophage T7 RNA polymerase" misc_feature 2865..2884 /note="#161 (N-terminal For)" misc_feature 2885..3231 /note="5'cytochrome c oxidase VIII (COX VIII) (LmxM.30)" CDS 3232..4032 /codon_start=1 /gene="aph(3')-II (or nptII)" /product="aminoglycoside phosphotransferase from Tn5" /note="NeoR/KanR" /note="confers resistance to neomycin, kanamycin, and G418 (Geneticin(R))" /translation="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE EHQGLAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" misc_feature complement(3371..3392) /note="reverse 519" misc_feature 4033..4601 /note="3'ribosomal protein L24, putative (LmxM.36)" misc_feature complement(4602..4621) /note="#162 (C-terminal Rev)" ORIGIN 1 ccagcttttg ttccctttag tgagggttaa ttgcgcgctt ggcgtaatca tggtcatagc 61 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca 121 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct 181 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac 241 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc 301 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt 361 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg 421 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg 481 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat 541 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta 601 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct 661 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc 721 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa 781 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg 841 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag 901 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt 961 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta 1021 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc 1081 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca 1141 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa 1201 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat 1261 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct 1321 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt 1381 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat 1441 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta 1501 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg 1561 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt 1621 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg 1681 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg 1741 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc 1801 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa 1861 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac 1921 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt 1981 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg 2041 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa 2101 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata 2161 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgcg ccctgtagcg 2221 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg 2281 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc 2341 cccgtcaagc tctaaatcgg gggcgccctt tagggttccg atttagtgct ttacggcacc 2401 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga 2461 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa 2521 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga 2581 tttcggccta ttggttaaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac 2641 aaaatattaa cgcttacaat ttccattcgc cattcaggct gcgcaactgt tgggaagggc 2701 gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc 2761 gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg 2821 agcgcgcgta atacgactca ctatagggcg aattggattt aaatgtataa tgcagacctg 2881 ctgctgacct acgcagcctt ttgcgcactt ggatctcctt ttgtcacgct tgagcaaaac 2941 gctgcacctc aagccgagca ttccctttcg tacgacagga acagccgggt ggtggtggtg 3001 gtagggggaa tggtgatgat cagatgcttc cgagatgctg aactctctcc ctctcgtaca 3061 tcctctgctt accctctcct tttttcttct ccatcggctg cattcatgct ttctccacaa 3121 cgcctttggc gtgacaagag ctcagtcgca cttgcgctcc tcttctcgcc tctgaaaagg 3181 tcctagctct cttctgtttt tttttgtaaa agcagctcat actcgaaggc tatgggatcg 3241 gccattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc 3301 ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca 3361 gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg 3421 caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg 3481 ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag 3541 gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg 3601 cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc 3661 atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa 3721 gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac 3781 ggcgatgatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat 3841 ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac 3901 atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc 3961 ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt 4021 gacgagttct tcagctccgc cggtgttcgt ccttctcgat gcatagtcca atgaccgtcc 4081 actgacgctg ccatccgtgt gcgatgtttc gagtgtactc tacctgccct tcgttgtgtt 4141 tctttttgtt ttcgtttttg agtaacgttt tcatgcaaag aaaaaaaaca aaaggagaag 4201 agccacgctc tcgtgtcgaa cttgtggaga atgggggaga ggctgaccac tgtctgtgcc 4261 gtgtgtgcat catgtacttc atgatgggca ccgttgatca tatatgagcg ttgtttcgcg 4321 cgtcgcatgg gcggttggtg cgctatcccg cacgacgtca cgcaccatgt agtgtttttt 4381 tttggtgttc gcctggtgtt tgttgcaatg ctccaacgtt tgcggatgcc cgtctctgtg 4441 acgctgagcc gtgctccatc tcgaaaagcg aagcctgcgc tggccgccaa ccaccttgcc 4501 ctacgtcgtg tatgcatctc acgttttctt ctcactgaca ttccatcgat acccctcccc 4561 ctcccttctt tgcttctgct gatggccgcg ctttctcatc tgcacaggtc tctcaaattg 4621 gatttaaat // LOCUS pTNeo 4629 bp ds-DNA circular SYN 16-MAY-2016 DEFINITION pTNeo ACCESSION . VERSION . KEYWORDS pTNeo SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4629) AUTHORS . TITLE Direct Submission JOURNAL Exported Tuesday, Jun 7, 2016 from SnapGene Viewer 3.1.2 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4629 /organism="synthetic DNA construct" /mol_type="other DNA" promoter complement(14..32) /note="T3 promoter" /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(53..69) /note="M13 rev" /note="common sequencing primer, one of multiple similar variants" protein_bind 77..93 /bound_moiety="lac repressor encoded by lacI" /note="lac operator" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." promoter complement(101..131) /note="lac promoter" /note="promoter for the E. coli lac operon" protein_bind 146..167 /bound_moiety="E. coli catabolite activator protein" /note="CAP binding site" /note="CAP binding activates transcription in the presence of cAMP." rep_origin complement(455..1043) /direction=LEFT /note="ori" /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(1214..2074) /codon_start=1 /gene="bla" /product="beta-lactamase" /note="AmpR" /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" promoter complement(2075..2179) /gene="bla" /note="AmpR promoter" rep_origin 2206..2662 /direction=RIGHT /note="f1 ori" /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind 2803..2819 /note="M13 fwd" /note="common sequencing primer, one of multiple similar variants" promoter 2829..2847 /note="T7 promoter" /note="promoter for bacteriophage T7 RNA polymerase" misc_feature 2865..2884 /note="#161 (N-terminal For)" misc_feature 2885..3231 /note="5'cytochrome c oxidase VIII (COX VIII) (LmxM.30)" CDS 3232..4032 /codon_start=1 /gene="aph(3')-II (or nptII)" /product="aminoglycoside phosphotransferase from Tn5" /note="NeoR/KanR" /note="confers resistance to neomycin, kanamycin, and G418 (Geneticin(R))" /translation="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE EHQGLAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" misc_feature complement(3371..3392) /note="reverse 519" misc_feature 4033..4601 /note="3'ribosomal protein L24, putative (LmxM.36)" misc_feature complement(4602..4621) /note="#162 (C-terminal Rev)" ORIGIN 1 ccagcttttg ttccctttag tgagggttaa ttgcgcgctt ggcgtaatca tggtcatagc 61 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca 121 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct 181 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac 241 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc 301 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt 361 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg 421 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg 481 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat 541 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta 601 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct 661 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc 721 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa 781 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg 841 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag 901 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt 961 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta 1021 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc 1081 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca 1141 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa 1201 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat 1261 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct 1321 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt 1381 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat 1441 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta 1501 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg 1561 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt 1621 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg 1681 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg 1741 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc 1801 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa 1861 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac 1921 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt 1981 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg 2041 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa 2101 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata 2161 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgcg ccctgtagcg 2221 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg 2281 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc 2341 cccgtcaagc tctaaatcgg gggcgccctt tagggttccg atttagtgct ttacggcacc 2401 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga 2461 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa 2521 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga 2581 tttcggccta ttggttaaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac 2641 aaaatattaa cgcttacaat ttccattcgc cattcaggct gcgcaactgt tgggaagggc 2701 gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc 2761 gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg 2821 agcgcgcgta atacgactca ctatagggcg aattggattt aaatgtataa tgcagacctg 2881 ctgctgacct acgcagcctt ttgcgcactt ggatctcctt ttgtcacgct tgagcaaaac 2941 gctgcacctc aagccgagca ttccctttcg tacgacagga acagccgggt ggtggtggtg 3001 gtagggggaa tggtgatgat cagatgcttc cgagatgctg aactctctcc ctctcgtaca 3061 tcctctgctt accctctcct tttttcttct ccatcggctg cattcatgct ttctccacaa 3121 cgcctttggc gtgacaagag ctcagtcgca cttgcgctcc tcttctcgcc tctgaaaagg 3181 tcctagctct cttctgtttt tttttgtaaa agcagctcat actcgaaggc tatgggatcg 3241 gccattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc 3301 ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca 3361 gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg 3421 caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg 3481 ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag 3541 gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg 3601 cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc 3661 atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa 3721 gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac 3781 ggcgatgatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat 3841 ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac 3901 atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc 3961 ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt 4021 gacgagttct tcagctccgc cggtgttcgt ccttctcgat gcatagtcca atgaccgtcc 4081 actgacgctg ccatccgtgt gcgatgtttc gagtgtactc tacctgccct tcgttgtgtt 4141 tctttttgtt ttcgtttttg agtaacgttt tcatgcaaag aaaaaaaaca aaaggagaag 4201 agccacgctc tcgtgtcgaa cttgtggaga atgggggaga ggctgaccac tgtctgtgcc 4261 gtgtgtgcat catgtacttc atgatgggca ccgttgatca tatatgagcg ttgtttcgcg 4321 cgtcgcatgg gcggttggtg cgctatcccg cacgacgtca cgcaccatgt agtgtttttt 4381 tttggtgttc gcctggtgtt tgttgcaatg ctccaacgtt tgcggatgcc cgtctctgtg 4441 acgctgagcc gtgctccatc tcgaaaagcg aagcctgcgc tggccgccaa ccaccttgcc 4501 ctacgtcgtg tatgcatctc acgttttctt ctcactgaca ttccatcgat acccctcccc 4561 ctcccttctt tgcttctgct gatggccgcg ctttctcatc tgcacaggtc tctcaaattg 4621 gatttaaat // LOCUS pTPuro 4726 bp ds-DNA circular SYN 28-APR-2016 DEFINITION pTPuro ACCESSION . VERSION . KEYWORDS pTPuro SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4726) AUTHORS . TITLE Direct Submission JOURNAL Exported Tuesday, Jun 7, 2016 from SnapGene Viewer 3.1.2 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4726 /organism="synthetic DNA construct" /mol_type="other DNA" promoter complement(14..32) /note="T3 promoter" /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(53..69) /note="M13 rev" /note="common sequencing primer, one of multiple similar variants" protein_bind 77..93 /bound_moiety="lac repressor encoded by lacI" /note="lac operator" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." promoter complement(101..131) /note="lac promoter" /note="promoter for the E. coli lac operon" protein_bind 146..167 /bound_moiety="E. coli catabolite activator protein" /note="CAP binding site" /note="CAP binding activates transcription in the presence of cAMP." rep_origin complement(455..1043) /direction=LEFT /note="ori" /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(1214..2074) /codon_start=1 /gene="bla" /product="beta-lactamase" /note="AmpR" /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" promoter complement(2075..2179) /gene="bla" /note="AmpR promoter" rep_origin 2206..2662 /direction=RIGHT /note="f1 ori" /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind 2803..2819 /note="M13 fwd" /note="common sequencing primer, one of multiple similar variants" promoter 2829..2847 /note="T7 promoter" /note="promoter for bacteriophage T7 RNA polymerase" misc_feature 2865..2884 /note="#161 (N-terminal For)" misc_feature 2885..3321 /note="5'malate dehydrogenase (LmxM.33)" CDS 3322..3921 /codon_start=1 /note="PuroR" /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA" misc_feature 3922..4698 /note="3'60S ribosomal protein L5, putative (LmxM.16)" misc_feature complement(4699..4718) /note="#162 (C-terminal Rev)" ORIGIN 1 ccagcttttg ttccctttag tgagggttaa ttgcgcgctt ggcgtaatca tggtcatagc 61 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca 121 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct 181 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac 241 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc 301 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt 361 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg 421 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg 481 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat 541 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta 601 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct 661 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc 721 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa 781 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg 841 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag 901 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt 961 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta 1021 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc 1081 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca 1141 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa 1201 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat 1261 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct 1321 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt 1381 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat 1441 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta 1501 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg 1561 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt 1621 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg 1681 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg 1741 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc 1801 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa 1861 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac 1921 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt 1981 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg 2041 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa 2101 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata 2161 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgcg ccctgtagcg 2221 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg 2281 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc 2341 cccgtcaagc tctaaatcgg gggcgccctt tagggttccg atttagtgct ttacggcacc 2401 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga 2461 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa 2521 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga 2581 tttcggccta ttggttaaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac 2641 aaaatattaa cgcttacaat ttccattcgc cattcaggct gcgcaactgt tgggaagggc 2701 gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc 2761 gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg 2821 agcgcgcgta atacgactca ctatagggcg aattggattt aaatgtataa tgcagacctg 2881 ctgcagccgt tgcattggtg gtcgtctctg ccgctgcccc ccccccttcc ccgtctatac 2941 gtgagtacgt tcgtgtgtgt gctatccgcg gcttcacaac gcttgcttgg tgcaagaggg 3001 ttgcggcgct gcgaccgtca taacacggcg gacaaggcgc acgcgcgcac accacagggt 3061 cacgacatat cacgagcgta atcgcatccg cacgacgcgg ccaacacact actctctctc 3121 tctgccgatc cccctccccc tttctctttg ctttcacatg tgcacctccg cctctacagg 3181 tttctctcac gaatctaagc gaaaacacag ttcaccgccg caactacatc gcagtcctcc 3241 ccgtgccgta caatccatct cgccttccca ctctgagcac atacacccac attcccacat 3301 tttcttttgc ggtctttcat aatgactgaa tacaagccaa cggttcgtct tgctacgcga 3361 gacgatgtgc cgcgggccgt acggacgctg gctgctgcgt tcgctgatta tcccgccacc 3421 cgacatacgg ttgacccaga tcgacacatt gagcgagtca cagaacttca agagttattc 3481 ttaacacggg tgggcttgga cattggtaag gtttgggttg cggacgatgg cgcggctgtt 3541 gcggtgtgga cgacgccaga gtcagtcgaa gcgggcgcag tgtttgctga gatcggtccc 3601 cgaatggcgg agcttagcgg gtcacgcctc gctgcacaac agcagatgga aggtctgttg 3661 gcaccacata gacctaagga gccggcttgg ttcttagcga cagttggcgt cagtccggac 3721 caccaaggta aggggctggg ttcagcggtt gttcttccag gagtagaggc tgcggaaagg 3781 gcaggtgtgc cggcgttcct tgaaacgtct gcaccacgta accttccttt ttacgagaga 3841 ttggggttta cagtaacagc agatgttgaa gtgcctgaag ggccgaggac atggtgcatg 3901 acacgtaagc cgggagccta agtcgagcgc tcacctctga atacgcgagt ctcttttggt 3961 ctcctgctct gacgcctctt ttctgattcc tgttcctttg gctgaaagaa gtgcatccgc 4021 atggttgtct tggaacaggg acagcgaggc gcgctggcgg caaccgcgtg cgcacgtgcc 4081 gctgagggaa cctcatatat ttcttttttt tttgttttca ttgctaattt tctgtgtgtc 4141 tttttctcgg cctcttgtcg cgtctgctcc ccctctctca ccactgatgg ggcgctagcg 4201 aggcggaaag gtcagagctt cctcgttata agtcattaga agggctgcac gggcacaccc 4261 tatgaaggag gaaggggtgc tgcgtggaaa agggagcgca gttggccaga acgcaagcgt 4321 gcgcgcttgt aggtgggcgc ttgcttgcta ctttttttct ctccgctctg ctctggctag 4381 cgatcttcgc cattgccgag tgggtgttgg cctgtctcag agactgcaac actgcttccc 4441 ggtacgggct gtgtgtggtc atcgcgtgtg cgcggctgca acgtgtgtag ggggaataca 4501 ggcacggtcc tccttcgatc tccgctgcca tggacgcgtc ctgtggaaga gcctgcctgc 4561 ggattgcctt tatgcgggcg ctcttccatc tctgcggcag cacaactttc tgcgcgtgtt 4621 gttcctctgt ttgttctttg gcatctccgc ttctgttggt gatgggtact gcctcgtgtc 4681 tccatgaact acaacgacgc acaggtctct caaattggat ttaaat //