LOCUS Exported 9154 bp ds-DNA circular SYN 24-MAR-2016 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pCas2F SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9154) AUTHORS . TITLE Direct Submission JOURNAL Exported Thursday, Mar 24, 2016 from SnapGene 3.1.1 http://www.snapgene.com COMMENT >pAQ1_YFP FEATURES Location/Qualifiers source 1..9154 /organism="synthetic DNA construct" /mol_type="other DNA" centromere 615..1475 /note="bet-lactamase" misc_feature 1630..2249 /note="pBR322 ori" misc_feature 2687..3225 /note="acsA up" misc_feature 3240..3343 /note="terminator" gene complement(3391..3427) /note="ssRa" gene complement(3428..4047) /note="tetR" RBS 4054..4065 /note="RBS" misc_feature 4074..4108 /note="J23113 Promoter" misc_feature 4110..4199 /note="Promoter" misc_feature 4130..4146 /note="TetO1" -10_signal 4147..4152 /note="-35" misc_feature 4153..4169 /note="TetO1" -10_signal 4170..4176 /note="-10" misc_feature 4177 /note="TSS from EKZ" CDS 4238..8341 /codon_start=1 /note="dCas9" /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL DATLIHQSITGLYETRIDLSQLGGD" misc_feature 4265..4267 /note="RuvC-like active site mutation" misc_feature 6755..6757 /note="HNH active site mutation" /note="HNH active site mutation (alive is CAC)" misc_feature 8383..8401 /note="T7 terminator" misc_feature 8448..8941 /note="acsA down" ORIGIN 1 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc 61 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag 121 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca 181 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc 241 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa 301 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct 361 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa 421 cgcttacaat ttcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg 481 catcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa 541 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt 601 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg 661 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag 721 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg 781 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg 841 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt 901 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga 961 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac 1021 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc 1081 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc 1141 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac 1201 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag 1261 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg 1321 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta 1381 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg 1441 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata 1501 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt 1561 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc 1621 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct 1681 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa 1741 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag 1801 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc 1861 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg 1921 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca 1981 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat 2041 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg 2101 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc 2161 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc 2221 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc 2281 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg 2341 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga 2401 gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc 2461 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa 2521 ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc 2581 gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg 2641 attacgccaa gctatttagg tgacactata gaatactcaa gctatgaggt gactgccgca 2701 ctcaatgttc aaccagatct agtgattatt tcggggttag cagccgatgc gggcaacctg 2761 gtgaagcaac tgcgagaatt aggttacaac ggcattattg ttggggggaa cggcttaaat 2821 acttctaata ttttccccgt ctgccaagca aaatgtgatg gggtgttggt ggcccaagcc 2881 tacagtgccg agttagataa tgagattaac cgcgcgtttc gggacgccta ttttcaacaa 2941 aaccaaaaag agccgcccca atttagtgcc caggctttta cggcgatcca agtttttgtt 3001 gaagccctca gcagcctcga tgaaaaaacg cccttagaaa ctcttgctct accggacttg 3061 cgacgacaac tgcgggacga aatttttgca ggtacctacg tcacgccttt gggtgaaatt 3121 tccttcacag aggaagggga aattgtccag aaggaatttt ttgtggccca aattgaaatg 3181 gatgaatcgg gtcaacaggg gcgtttcgcc ttcattgaaa cgaactataa acgcagaaag 3241 gcccacccga aggtgagcca gtgtgactct agtagagagc gttcaccgac aaacaacaga 3301 taaaacgaaa ggcccagtct ttcgactgag cctttcgttt tatttgatgc ctggctctag 3361 tagtgatcta cactagcact atcagtgtta ttaagctact aaagcgtagt tttcgtcgtt 3421 tgcagcggac ccactttcac atttaagttg tttttctaat ccgcatatga tcaattcaag 3481 gccgaataag aaggctggct ctgcaccttg gtgatcaaat aattcgatag cttgtcgtaa 3541 taatggcggc atactatcag tagtaggtgt ttccctttct tctttagcga cttgatgctc 3601 ttgatcttcc aatacgcaac ctaaagtaaa atgccccaca gcgctgagtg catataatgc 3661 attctctagt gaaaaacctt gttggcataa aaaggctaat tgattttcga gagtttcata 3721 ctgtttttct gtaggccgtg tacctaaatg tacttttgct ccatcgcgat gacttagtaa 3781 agcacatcta aaacttttag cgttattacg taaaaaatct tgccagcttt ccccttctaa 3841 agggcaaaag tgagtatggt gcctatctaa catctcaatg gctaaggcgt cgagcaaagc 3901 ccgcttattt tttacatgcc aatacaatgt aggctgctct acacctagct tctgggcgag 3961 tttacgggtt gttaaacctt cgattccgac ctcattaagc agctctaatg cgctgttaat 4021 cactttactt ttatctaatc tggacatcta gtatttctcc tctttctcta gtagctagca 4081 taatccctag gactgagcta gccatcaggt taacaaaaaa gcaggaatat ccctatcagt 4141 gatagacaga catccctatc agtgatagag tataaagtta actgtgggat tgcaaaagga 4201 gctcccaact cataaagtca agttgggcgt taattccatg gataagaaat actcaatagg 4261 cttagctatc ggcacaaata gcgtcggatg ggcggtgatc actgatgaat ataaggttcc 4321 gtctaaaaag ttcaaggttc tgggaaatac agaccgccac agtatcaaaa aaaatcttat 4381 aggggctctt ttatttgaca gtggagagac agcggaagcg actcgtctca aacggacagc 4441 tcgtagaagg tatacacgtc ggaagaatcg tatttgttat ctacaggaga ttttttcaaa 4501 tgagatggcg aaagtagatg atagtttctt tcatcgactt gaagagtctt ttttggtgga 4561 agaagacaag aagcatgaac gtcatcctat ttttggaaat atagtagatg aagttgctta 4621 tcatgagaaa tatccaacta tctatcatct gcgaaaaaaa ttggtagatt ctactgataa 4681 agcggatttg cgcttaatct atttggcctt agcgcatatg attaagtttc gtggtcattt 4741 tttgattgag ggagatttaa atcctgataa tagtgatgtg gacaaactat ttatccagtt 4801 ggtacaaacc tacaatcaat tatttgaaga aaaccctatt aacgcaagtg gagtagatgc 4861 taaagcgatt ctttctgcac gattgagtaa atcaagacga ttagaaaatc tcattgctca 4921 gctccccggt gagaagaaaa atggattatt tgggaatctc attgctttgt cattgggttt 4981 gacccctaat tttaaatcaa attttgattt ggcagaagat gctaaattac agctttcaaa 5041 agatacttac gatgatgatt tagataattt attggcgcaa attggagatc aatatgctga 5101 tttgtttttg gcagctaaga atttatcaga tgctatttta ctttcagata tcctaagagt 5161 aaatactgaa ataactaagg ctcccctatc agcttcaatg attaaacgct acgatgaaca 5221 tcatcaagac ttgactcttt taaaagcttt agttcgacaa caacttccag aaaagtataa 5281 agaaatcttt tttgatcaat caaaaaacgg atatgcaggt tatattgatg ggggagctag 5341 ccaagaagaa ttttataaat ttatcaaacc aattttagaa aaaatggatg gtactgagga 5401 attattggtg aaactaaatc gtgaagattt gctgcgcaag caacggacct ttgacaacgg 5461 ctctattccc catcaaattc acttgggtga gctgcatgct attttgagaa gacaagaaga 5521 cttttatcca tttttaaaag acaatcgtga gaagattgaa aaaatcttga cttttcgaat 5581 tccttattat gttggtccat tggcgcgtgg caatagtcgt tttgcatgga tgactcggaa 5641 gtctgaagaa acaattaccc catggaattt tgaagaagtt gtcgataaag gtgcttcagc 5701 tcaatcattt attgaacgca tgacaaactt tgataaaaat cttccaaatg aaaaagtact 5761 accaaaacat agtttgcttt atgagtattt tacggtttat aacgaattga caaaggtcaa 5821 atatgttact gaaggaatgc gaaaaccagc atttctttca ggtgaacaga agaaagccat 5881 tgttgattta ctcttcaaaa caaatcgaaa agtaaccgtt aagcaattaa aagaagatta 5941 tttcaaaaaa atagaatgtt ttgatagtgt tgaaatttca ggagttgaag atagatttaa 6001 tgcttcatta ggtacctacc atgatttgct aaaaattatt aaagataaag attttttgga 6061 taatgaagaa aatgaagata tcttagagga tattgtttta acattgacct tatttgaaga 6121 tagggagatg attgaggaaa gacttaaaac atatgctcac ctctttgatg ataaggtgat 6181 gaaacagctt aaacgtcgcc gttatactgg ttggggacgt ttgtctcgaa aattgattaa 6241 tggtattagg gataagcaat ctggcaaaac aatattagat tttttgaaat cagatggttt 6301 tgccaatcgc aattttatgc agctgatcca tgatgatagt ttgacattta aagaagacat 6361 tcaaaaagca caagtgtctg gacaaggcga tagtttacat gaacatattg caaatttagc 6421 tggtagccct gctattaaaa aaggtatttt acagactgta aaagttgttg atgaattggt 6481 caaagtaatg gggcggcata agccagaaaa tatcgttatt gaaatggcac gtgaaaatca 6541 gacaactcaa aagggccaga aaaattcgcg agagcgtatg aaacgaatcg aagaaggtat 6601 caaagaatta ggaagtcaga ttcttaaaga gcatcctgtt gaaaatactc aattgcaaaa 6661 tgaaaagctc tatctctatt atctccaaaa tggaagagac atgtatgtgg accaagaatt 6721 agatattaat cgtttaagtg attatgatgt cgatgccatt gttccacaaa gtttccttaa 6781 agacgattca atagacaata aggtcttaac gcgttctgat aaaaatcgtg gtaaatcgga 6841 taacgttcca agtgaagaag tagtcaaaaa gatgaaaaac tattggagac aacttctaaa 6901 cgccaagtta atcactcaac gtaagtttga taatttaacg aaagctgaac gtggaggttt 6961 gagtgaactt gataaagctg gttttatcaa acgccaattg gttgaaactc gccaaatcac 7021 taagcatgtg gcacaaattt tggatagtcg catgaatact aaatacgatg aaaatgataa 7081 acttattcga gaggttaaag tgattacctt aaaatctaaa ttagtttctg acttccgaaa 7141 agatttccaa ttctataaag tacgtgagat taacaattac catcatgccc atgatgcgta 7201 tctaaatgcc gtcgttggaa ctgctttgat taagaaatat ccaaaacttg aatcggagtt 7261 tgtctatggt gattataaag tttatgatgt tcgtaaaatg attgctaagt ctgagcaaga 7321 aataggcaaa gcaaccgcaa aatatttctt ttactctaat atcatgaact tcttcaaaac 7381 agaaattaca cttgcaaatg gagagattcg caaacgccct ctaatcgaaa ctaatgggga 7441 aactggagaa attgtctggg ataaagggcg agattttgcc acagtgcgca aagtattgtc 7501 catgccccaa gtcaatattg tcaagaaaac agaagtacag acaggcggat tctccaagga 7561 gtcaatttta ccaaaaagaa attcggacaa gcttattgct cgtaaaaaag actgggatcc 7621 aaaaaaatat ggtggttttg atagtccaac ggtagcttat tcagtcctag tggttgctaa 7681 ggtggaaaaa gggaaatcga agaagttaaa atccgttaaa gagttactag ggatcacaat 7741 tatggaaaga agttcctttg aaaaaaatcc gattgacttt ttagaagcta aaggatataa 7801 ggaagttaaa aaagacttaa tcattaaact acctaaatat agtctttttg agttagaaaa 7861 cggtcgtaaa cggatgctgg ctagtgccgg agaattacaa aaaggaaatg agctggctct 7921 gccaagcaaa tatgtgaatt ttttatattt agctagtcat tatgaaaagt tgaagggtag 7981 tccagaagat aacgaacaaa aacaattgtt tgtggagcag cataagcatt atttagatga 8041 gattattgag caaatcagtg aattttctaa gcgtgttatt ttagcagatg ccaatttaga 8101 taaagttctt agtgcatata acaaacatag agacaaacca atacgtgaac aagcagaaaa 8161 tattattcat ttatttacgt tgacgaatct tggagctccc gctgctttta aatattttga 8221 tacaacaatt gatcgtaaac gatatacgtc tacaaaagaa gttttagatg ccactcttat 8281 ccatcaatcc atcactggtc tttatgaaac acgcattgat ttgagtcagc taggaggtga 8341 ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa taactagcat 8401 aaccccttgg ggcctctaaa cgggtcttga cgggtttttt gtctagagcg ctcaagttga 8461 tcgaatgtat gccccggata tgacctaggg agattcttct aaagcgagca gggtttgcca 8521 agtggcgatc gcctcaggga accataacca actttgttcg aggcggcggg ggttgagctg 8581 ggaacgatcg agatctaggg cgatcgcccg gagtttttcg gctttcccta caagaatttc 8641 ctggtcaacg tcggtgggtt gcctttgggc caaatcgtac atactaatcg ccaggcccgc 8701 ataaatctcc gcctggaggg ctggggggaa atcctgggtc gttgccgccg ctaaggcttc 8761 ataccaacga tcattggcgg cttcgtaatc tgcattgagg taatggacaa aacccagggc 8821 catcaaaatt tcggggtcgt tgggctgttg ggctagggcc atgtcccaat tgtcttgcac 8881 cgtggcgatc gccgttgctt gggttggcga tggtgggacg gcagccaaag ttgtttgcca 8941 gcatgcgccc tatagtgagt cgtattacaa ttcactggcc gtcgttttac aacgtcgtga 9001 ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag 9061 ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa 9121 tggcgaatgg acgcgccctg tagcggcgca ttaa //