LOCUS Exported 9326 bp ds-DNA circular SYN 16-JUN-2022 DEFINITION Expresses the tracrRNA, the dCas9 catalytic site mutant and a CRISPR array designed for the easy cloning of new spacers.. ACCESSION . VERSION . KEYWORDS pdCas9 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9326) AUTHORS Bikard D, Jiang W, Samai P, Hochschild A, Zhang F, Marraffini LA TITLE Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system. JOURNAL Nucleic Acids Res. 2013 Jun 12. PUBMED 23761437 REFERENCE 2 (bases 1 to 9326) AUTHORS . TITLE Direct Submission JOURNAL Exported Jun 16, 2022 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..9326 /organism="synthetic DNA construct" /mol_type="other DNA" misc_feature 2605..2736 /label=crRNA leader /note="crRNA leader sequence for the Streptococcus pyogenes CRISPR/Cas system" repeat_region 2737..2772 /label=DR /note="direct repeat for the Streptococcus pyogenes CRISPR/Cas system" repeat_region 2803..2838 /label=DR /note="direct repeat for the Streptococcus pyogenes CRISPR/Cas system" primer_bind complement(3267..3286) /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" CDS complement(5139..5798) /codon_start=1 /gene="cat" /product="chloramphenicol acetyltransferase" /label=CmR /note="confers resistance to chloramphenicol" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA" primer_bind 5713..5732 /label=CAT-R /note="Chloramphenicol resistance gene, reverse primer" promoter complement(5799..5901) /label=cat promoter /note="promoter of the E. coli cat gene encoding chloramphenicol acetyltransferase" primer_bind complement(6293..6310) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(6427..6972) /direction=LEFT /label=p15A ori /note="Plasmids containing the medium-copy-number p15A origin of replication can be propagated in E. coli cells that contain a second plasmid with the ColE1 origin. " primer_bind complement(6507..6526) /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 7084..7112 /label=tet promoter /note="E. coli promoter for tetracycline efflux protein gene" misc_RNA complement(7430..7508) /label=tracrRNA /note="trans-activating CRISPR RNA for the Streptococcus pyogenes CRISPR/Cas9 system" CDS 7804..2584 /codon_start=1 /product="catalytically dead mutant of the Cas9 endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /label=dCas9 /note="RNA-guided DNA-binding protein that lacks endonuclease activity due to the D10A mutation in the RuvC catalytic domain and the H840A mutation in the HNH catalytic domain" /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" ORIGIN 1 accaaaacat agtttgcttt atgagtattt tacggtttat aacgaattga caaaggtcaa 61 atatgttact gaaggaatgc gaaaaccagc atttctttca ggtgaacaga agaaagccat 121 tgttgattta ctcttcaaaa caaatcgaaa agtaaccgtt aagcaattaa aagaagatta 181 tttcaaaaaa atagaatgtt ttgatagtgt tgaaatttca ggagttgaag atagatttaa 241 tgcttcatta ggtacctacc atgatttgct aaaaattatt aaagataaag attttttgga 301 taatgaagaa aatgaagata tcttagagga tattgtttta acattgacct tatttgaaga 361 tagggagatg attgaggaaa gacttaaaac atatgctcac ctctttgatg ataaggtgat 421 gaaacagctt aaacgtcgcc gttatactgg ttggggacgt ttgtctcgaa aattgattaa 481 tggtattagg gataagcaat ctggcaaaac aatattagat tttttgaaat cagatggttt 541 tgccaatcgc aattttatgc agctgatcca tgatgatagt ttgacattta aagaagacat 601 tcaaaaagca caagtgtctg gacaaggcga tagtttacat gaacatattg caaatttagc 661 tggtagccct gctattaaaa aaggtatttt acagactgta aaagttgttg atgaattggt 721 caaagtaatg gggcggcata agccagaaaa tatcgttatt gaaatggcac gtgaaaatca 781 gacaactcaa aagggccaga aaaattcgcg agagcgtatg aaacgaatcg aagaaggtat 841 caaagaatta ggaagtcaga ttcttaaaga gcatcctgtt gaaaatactc aattgcaaaa 901 tgaaaagctc tatctctatt atctccaaaa tggaagagac atgtatgtgg accaagaatt 961 agatattaat cgtttaagtg attatgatgt cgatgccatt gttccacaaa gtttccttaa 1021 agacgattca atagacaata aggtcttaac gcgttctgat aaaaatcgtg gtaaatcgga 1081 taacgttcca agtgaagaag tagtcaaaaa gatgaaaaac tattggagac aacttctaaa 1141 cgccaagtta atcactcaac gtaagtttga taatttaacg aaagctgaac gtggaggttt 1201 gagtgaactt gataaagctg gttttatcaa acgccaattg gttgaaactc gccaaatcac 1261 taagcatgtg gcacaaattt tggatagtcg catgaatact aaatacgatg aaaatgataa 1321 acttattcga gaggttaaag tgattacctt aaaatctaaa ttagtttctg acttccgaaa 1381 agatttccaa ttctataaag tacgtgagat taacaattac catcatgccc atgatgcgta 1441 tctaaatgcc gtcgttggaa ctgctttgat taagaaatat ccaaaacttg aatcggagtt 1501 tgtctatggt gattataaag tttatgatgt tcgtaaaatg attgctaagt ctgagcaaga 1561 aataggcaaa gcaaccgcaa aatatttctt ttactctaat atcatgaact tcttcaaaac 1621 agaaattaca cttgcaaatg gagagattcg caaacgccct ctaatcgaaa ctaatgggga 1681 aactggagaa attgtctggg ataaagggcg agattttgcc acagtgcgca aagtattgtc 1741 catgccccaa gtcaatattg tcaagaaaac agaagtacag acaggcggat tctccaagga 1801 gtcaatttta ccaaaaagaa attcggacaa gcttattgct cgtaaaaaag actgggatcc 1861 aaaaaaatat ggtggttttg atagtccaac ggtagcttat tcagtcctag tggttgctaa 1921 ggtggaaaaa gggaaatcga agaagttaaa atccgttaaa gagttactag ggatcacaat 1981 tatggaaaga agttcctttg aaaaaaatcc gattgacttt ttagaagcta aaggatataa 2041 ggaagttaaa aaagacttaa tcattaaact acctaaatat agtctttttg agttagaaaa 2101 cggtcgtaaa cggatgctgg ctagtgccgg agaattacaa aaaggaaatg agctggctct 2161 gccaagcaaa tatgtgaatt ttttatattt agctagtcat tatgaaaagt tgaagggtag 2221 tccagaagat aacgaacaaa aacaattgtt tgtggagcag cataagcatt atttagatga 2281 gattattgag caaatcagtg aattttctaa gcgtgttatt ttagcagatg ccaatttaga 2341 taaagttctt agtgcatata acaaacatag agacaaacca atacgtgaac aagcagaaaa 2401 tattattcat ttatttacgt tgacgaatct tggagctccc gctgctttta aatattttga 2461 tacaacaatt gatcgtaaac gatatacgtc tacaaaagaa gttttagatg ccactcttat 2521 ccatcaatcc atcactggtc tttatgaaac acgcattgat ttgagtcagc taggaggtga 2581 ctgaagtata ttttagatga agattatttc ttaataacta aaaatatggt ataatactct 2641 taataaatgc agtaatacag gggcttttca agactgaagt ctagctgaga caaatagtgc 2701 gattacgaaa ttttttagac aaaaatagtc tacgaggttt tagagctatg ctgttttgaa 2761 tggtcccaaa actgagacca gtctcggaag ctcaaaggtc tcgttttaga gctatgctgt 2821 tttgaatggt cccaaaactt cagcacactg agacttgttg agttccatgt tttagagcta 2881 tgctgttttg aatggactcc attcaacatt gccgatgata acttgagaaa gagggttaat 2941 accagcagtc ggataccttc ctattctttc tgttaaagcg ttttcatgtt ataataggca 3001 aaagaagagt agtgtgatcg tccattccga cagcatcgcc agtcactatg gcgtgctgct 3061 agcgctatat gcgttgatgc aatttctatg cgcacccgtt ctcggagcac tgtccgaccg 3121 ctttggccgc cgcccagtcc tgctcgcttc gctacttgga gccactatcg actacgcgat 3181 catggcgacc acacccgtcc tgtggatcct ctacgccgga cgcatcgtgg ccggcatcac 3241 cggcgccaca ggtgcggttg ctggcgccta tatcgccgac atcaccgatg gggaagatcg 3301 ggctcgccac ttcgggctca tgagcgcttg tttcggcgtg ggtatggtgg caggccccgt 3361 ggccggggga ctgttgggcg ccatctcctt gcatgcacca ttccttgcgg cggcggtgct 3421 caacggcctc aacctactac tgggctgctt cctaatgcag gagtcgcata agggagagcg 3481 tcgaccgatg cccttgagag ccttcaaccc agtcagctcc ttccggtggg cgcggggcat 3541 gactatcgtc gccgcactta tgactgtctt ctttatcatg caactcgtag gacaggtgcc 3601 ggcagcgctc tgggtcattt tcggcgagga ccgctttcgc tggagcgcga cgatgatcgg 3661 cctgtcgctt gcggtattcg gaatcttgca cgccctcgct caagccttcg tcactggtcc 3721 cgccaccaaa cgtttcggcg agaagcaggc cattatcgcc ggcatggcgg ccgacgcgct 3781 gggctacgtc ttgctggcgt tcgcgacgcg aggctggatg gccttcccca ttatgattct 3841 tctcgcttcc ggcggcatcg ggatgcccgc gttgcaggcc atgctgtcca ggcaggtaga 3901 tgacgaccat cagggacagc ttcaaggatc gctcgcggct cttaccagcc taacttcgat 3961 cattggaccg ctgatcgtca cggcgattta tgccgcctcg gcgagcacat ggaacgggtt 4021 ggcatggatt gtaggcgccg ccctatacct tgtctgcctc cccgcgttgc gtcgcggtgc 4081 atggagccgg gccacctcga cctgaatgga agccggcggc acctcgctaa cggattcacc 4141 actccaagaa ttggagccaa tcaattcttg cggagaactg tgaatgcgca aaccaaccct 4201 tggcagaaca tatccatcgc gtccgccatc tccagcagcc gcacgcggcg catctcgggc 4261 agcgttgggt cctggccacg ggtgcgcatg atcgtgctcc tgtcgttgag gacccggcta 4321 ggctggcggg gttgccttac tggttagcag aatgaatcac cgatacgcga gcgaacgtga 4381 agcgactgct gctgcaaaac gtctgcgacc tgagcaacaa catgaatggt cttcggtttc 4441 cgtgtttcgt aaagtctgga aacgcggaag tcccctacgt gctgctgaag ttgcccgcaa 4501 cagagagtgg aaccaaccgg tgataccacg atactatgac tgagagtcaa cgccatgagc 4561 ggcctcattt cttattctga gttacaacag tccgcaccgc tgccggtagc tccttccggt 4621 gggcgcgggg catgactatc gtcgccgcac ttatgactgt cttctttatc atgcaactcg 4681 taggacaggt gccggcagcg cccaacagtc ccccggccac ggggcctgcc accataccca 4741 cgccgaaaca agcgccctgc accattatgt tccggatctg catcgcagga tgctgctggc 4801 taccctgtgg aacacctaca tctgtattaa cgaagcgcta accgttttta tcaggctctg 4861 ggaggcagaa taaatgatca tatcgtcaat tattacctcc acggggagag cctgagcaaa 4921 ctggcctcag gcatttgaga agcacacggt cacactgctt ccggtagtca ataaaccggt 4981 aaaccagcaa tagacataag cggctattta acgaccctgc cctgaaccga cgaccgggtc 5041 gaatttgctt tcgaatttct gccattcatc cgcttattat cacttattca ggcgtagcaa 5101 ccaggcgttt aagggcacca ataactgcct taaaaaaatt acgccccgcc ctgccactca 5161 tcgcagtact gttgtaattc attaagcatt ctgccgacat ggaagccatc acaaacggca 5221 tgatgaacct gaatcgccag cggcatcagc accttgtcgc cttgcgtata atatttgccc 5281 atggtgaaaa cgggggcgaa gaagttgtcc atattggcca cgtttaaatc aaaactggtg 5341 aaactcaccc agggattggc tgagacgaaa aacatattct caataaaccc tttagggaaa 5401 taggccaggt tttcaccgta acacgccaca tcttgcgaat atatgtgtag aaactgccgg 5461 aaatcgtcgt ggtattcact ccagagcgat gaaaacgttt cagtttgctc atggaaaacg 5521 gtgtaacaag ggtgaacact atcccatatc accagctcac cgtctttcat tgccatacgg 5581 aattccggat gagcattcat caggcgggca agaatgtgaa taaaggccgg ataaaacttg 5641 tgcttatttt tctttacggt ctttaaaaag gccgtaatat ccagctgaac ggtctggtta 5701 taggtacatt gagcaactga ctgaaatgcc tcaaaatgtt ctttacgatg ccattgggat 5761 atatcaacgg tggtatatcc agtgattttt ttctccattt tagcttcctt agctcctgaa 5821 aatctcgata actcaaaaaa tacgcccggt agtgatctta tttcattatg gtgaaagttg 5881 gaacctctta cgtgccgatc aacgtctcat tttcgccaaa agttggcccg gggcttcccg 5941 gtatcaacag ggacaccagg atttatttat tctgcgaagt gatcttccgt cacaggtatt 6001 tattcggcgc aaagtgcgtc gggtgatgct gccaacttac tgatttagtg tatgatggtg 6061 tttttgaggt gctccagtgg cttctgtttc tatcagctgt ccctcctgtt cagctactga 6121 cggggtggtg cgtaacggca aaagcaccgc cggacatcag cgctagcgga gtgtatactg 6181 gcttactatg ttggcactga tgagggtgtc agtgaagtgc ttcatgtggc aggagaaaaa 6241 aggctgcacc ggtgcgtcag cagaatatgt gatacaggat atattccgct tcctcgctca 6301 ctgactcgct acgctcggtc gttcgactgc ggcgagcgga aatggcttac gaacggggcg 6361 gagatttcct ggaagatgcc aggaagatac ttaacaggga agtgagaggg ccgcggcaaa 6421 gccgtttttc cataggctcc gcccccctga caagcatcac gaaatctgac gctcaaatca 6481 gtggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gcggctccct 6541 cgtgcgctct cctgttcctg cctttcggtt taccggtgtc attccgctgt tatggccgcg 6601 tttgtctcat tccacgcctg acactcagtt ccgggtaggc agttcgctcc aagctggact 6661 gtatgcacga accccccgtt cagtccgacc gctgcgcctt atccggtaac tatcgtcttg 6721 agtccaaccc ggaaagacat gcaaaagcac cactggcagc agccactggt aattgattta 6781 gaggagttag tcttgaagtc atgcgccggt taaggctaaa ctgaaaggac aagttttggt 6841 gactgcgctc ctccaagcca gttacctcgg ttcaaagagt tggtagctca gagaaccttc 6901 gaaaaaccgc cctgcaaggc ggttttttcg ttttcagagc aagagattac gcgcagacca 6961 aaacgatctc aagaagatca tcttattaat cagataaaat atttctagat ttcagtgcaa 7021 tttatctctt caaatgtagc acctgaagtc agccccatac gatataagtt gtaattctaa 7081 tgtttgacag cttatcatcg ataagcttta atgcggtagt ttatcacagt taaattgcta 7141 acgcagtcag gcaccgtgta tgaaatctaa caatgcgctc atcgtcatcc tcggcaccgt 7201 caccctggat gctgtaggca taggcttggt tatgccggta ctgccgggcc tcttgcggga 7261 ttacgaaatc atcctgtgga gcttagtagg tttagcaaga tggcagcgcc taaatgtaga 7321 atgataaaag gattaagaga ttaatttccc taaaaatgat aaaacaagcg ttttgaaagc 7381 gcttgttttt ttggtttgca gtcagagtag aatagaagta tcaaaaaaag caccgactcg 7441 gtgccacttt ttcaagttga taacggacta gccttatttt aacttgctat gctgttttga 7501 atggttccaa caagattatt ttataacttt tataacaaat aatcaaggag aaattcaaag 7561 aaatttatca gccataaaac aatacttaat actatagaat gataacaaaa taaactactt 7621 tttaaaagaa ttttgtgtta taatctattt attattaagt attgggtaat attttttgaa 7681 gagatatttt gaaaaagaaa aattaaagca tattaaacta atttcggagg tcattaaaac 7741 tattattgaa atcatcaaac tcattatgga tttaatttaa actttttatt ttaggaggca 7801 aaaatggata agaaatactc aataggctta gctatcggca caaatagcgt cggatgggcg 7861 gtgatcactg atgaatataa ggttccgtct aaaaagttca aggttctggg aaatacagac 7921 cgccacagta tcaaaaaaaa tcttataggg gctcttttat ttgacagtgg agagacagcg 7981 gaagcgactc gtctcaaacg gacagctcgt agaaggtata cacgtcggaa gaatcgtatt 8041 tgttatctac aggagatttt ttcaaatgag atggcgaaag tagatgatag tttctttcat 8101 cgacttgaag agtctttttt ggtggaagaa gacaagaagc atgaacgtca tcctattttt 8161 ggaaatatag tagatgaagt tgcttatcat gagaaatatc caactatcta tcatctgcga 8221 aaaaaattgg tagattctac tgataaagcg gatttgcgct taatctattt ggccttagcg 8281 catatgatta agtttcgtgg tcattttttg attgagggag atttaaatcc tgataatagt 8341 gatgtggaca aactatttat ccagttggta caaacctaca atcaattatt tgaagaaaac 8401 cctattaacg caagtggagt agatgctaaa gcgattcttt ctgcacgatt gagtaaatca 8461 agacgattag aaaatctcat tgctcagctc cccggtgaga agaaaaatgg cttatttggg 8521 aatctcattg ctttgtcatt gggtttgacc cctaatttta aatcaaattt tgatttggca 8581 gaagatgcta aattacagct ttcaaaagat acttacgatg atgatttaga taatttattg 8641 gcgcaaattg gagatcaata tgctgatttg tttttggcag ctaagaattt atcagatgct 8701 attttacttt cagatatcct aagagtaaat actgaaataa ctaaggctcc cctatcagct 8761 tcaatgatta aacgctacga tgaacatcat caagacttga ctcttttaaa agctttagtt 8821 cgacaacaac ttccagaaaa gtataaagaa atcttttttg atcaatcaaa aaacggatat 8881 gcaggttata ttgatggggg agctagccaa gaagaatttt ataaatttat caaaccaatt 8941 ttagaaaaaa tggatggtac tgaggaatta ttggtgaaac taaatcgtga agatttgctg 9001 cgcaagcaac ggacctttga caacggctct attccccatc aaattcactt gggtgagctg 9061 catgctattt tgagaagaca agaagacttt tatccatttt taaaagacaa tcgtgagaag 9121 attgaaaaaa tcttgacttt tcgaattcct tattatgttg gtccattggc gcgtggcaat 9181 agtcgttttg catggatgac tcggaagtct gaagaaacaa ttaccccatg gaattttgaa 9241 gaagttgtcg ataaaggtgc ttcagctcaa tcatttattg aacgcatgac aaactttgat 9301 aaaaatcttc caaatgaaaa agtact //