LOCUS Exported 9326 bp ds-DNA circular SYN 17-JUN-2022 DEFINITION Bacterial expression of Cas9 nuclease, tracrRNA and crRNA guide. ACCESSION . VERSION . KEYWORDS pCas9 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9326) AUTHORS Jiang W, Bikard D, Cox D, Zhang F, Marraffini LA TITLE RNA-guided editing of bacterial genomes using CRISPR-Cas systems. JOURNAL Nat Biotechnol. 2013 Jan 29. doi: 10.1038/nbt.2508. PUBMED 23360965 REFERENCE 2 (bases 1 to 9326) AUTHORS . TITLE Direct Submission JOURNAL Exported Jun 17, 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_RNA 138..216 /label=tracrRNA /note="trans-activating CRISPR RNA for the Streptococcus pyogenes CRISPR/Cas9 system" promoter complement(534..562) /label=tet promoter /note="E. coli promoter for tetracycline efflux protein gene" rep_origin 674..1219 /direction=RIGHT /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 1120..1139 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 1336..1353 /label=L4440 /note="L4440 vector, forward primer" promoter 1745..1847 /label=cat promoter /note="promoter of the E. coli cat gene encoding chloramphenicol acetyltransferase" CDS 1848..2507 /codon_start=1 /gene="cat" /product="chloramphenicol acetyltransferase" /label=CmR /note="confers resistance to chloramphenicol" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA" primer_bind complement(1914..1933) /label=CAT-R /note="Chloramphenicol resistance gene, reverse primer" primer_bind 4360..4379 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" repeat_region 4808..4843 /label=DR /note="direct repeat for the Streptococcus pyogenes CRISPR/Cas system" repeat_region 4874..4909 /label=DR /note="direct repeat for the Streptococcus pyogenes CRISPR/Cas system" misc_feature complement(4910..5041) /label=crRNA leader /note="crRNA leader sequence for the Streptococcus pyogenes CRISPR/Cas system" CDS complement(5062..9168) /codon_start=1 /product="Cas9 (Csn1) endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /label=Cas9 /note="generates RNA-guided double strand breaks in DNA" /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL DATLIHQSITGLYETRIDLSQLGGD" ORIGIN 1 gattataaca caaaattctt ttaaaaagta gtttattttg ttatcattct atagtattaa 61 gtattgtttt atggctgata aatttctttg aatttctcct tgattatttg ttataaaagt 121 tataaaataa tcttgttgga accattcaaa acagcatagc aagttaaaat aaggctagtc 181 cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt tttgatactt ctattctact 241 ctgactgcaa accaaaaaaa caagcgcttt caaaacgctt gttttatcat ttttagggaa 301 attaatctct taatcctttt atcattctac atttaggcgc tgccatcttg ctaaacctac 361 taagctccac aggatgattt cgtaatcccg caagaggccc ggcagtaccg gcataaccaa 421 gcctatgcct acagcatcca gggtgacggt gccgaggatg acgatgagcg cattgttaga 481 tttcatacac ggtgcctgac tgcgttagca atttaactgt gataaactac cgcattaaag 541 cttatcgatg ataagctgtc aaacatgaga attacaactt atatcgtatg gggctgactt 601 caggtgctac atttgaagag ataaattgca ctgaaatcta gaaatatttt atctgattaa 661 taagatgatc ttcttgagat cgttttggtc tgcgcgtaat ctcttgctct gaaaacgaaa 721 aaaccgcctt gcagggcggt ttttcgaagg ttctctgagc taccaactct ttgaaccgag 781 gtaactggct tggaggagcg cagtcaccaa aacttgtcct ttcagtttag ccttaaccgg 841 cgcatgactt caagactaac tcctctaaat caattaccag tggctgctgc cagtggtgct 901 tttgcatgtc tttccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg 961 gactgaacgg ggggttcgtg catacagtcc agcttggagc gaactgccta cccggaactg 1021 agtgtcaggc gtggaatgag acaaacgcgg ccataacagc ggaatgacac cggtaaaccg 1081 aaaggcagga acaggagagc gcacgaggga gccgccaggg ggaaacgcct ggtatcttta 1141 tagtcctgtc gggtttcgcc accactgatt tgagcgtcag atttcgtgat gcttgtcagg 1201 ggggcggagc ctatggaaaa acggctttgc cgcggccctc tcacttccct gttaagtatc 1261 ttcctggcat cttccaggaa atctccgccc cgttcgtaag ccatttccgc tcgccgcagt 1321 cgaacgaccg agcgtagcga gtcagtgagc gaggaagcgg aatatatcct gtatcacata 1381 ttctgctgac gcaccggtgc agcctttttt ctcctgccac atgaagcact tcactgacac 1441 cctcatcagt gccaacatag taagccagta tacactccgc tagcgctgat gtccggcggt 1501 gcttttgccg ttacgcacca ccccgtcagt agctgaacag gagggacagc tgatagaaac 1561 agaagccact ggagcacctc aaaaacacca tcatacacta aatcagtaag ttggcagcat 1621 cacccgacgc actttgcgcc gaataaatac ctgtgacgga agatcacttc gcagaataaa 1681 taaatcctgg tgtccctgtt gataccggga agccccgggc caacttttgg cgaaaatgag 1741 acgttgatcg gcacgtaaga ggttccaact ttcaccataa tgaaataaga tcactaccgg 1801 gcgtattttt tgagttatcg agattttcag gagctaagga agctaaaatg gagaaaaaaa 1861 tcactggata taccaccgtt gatatatccc aatggcatcg taaagaacat tttgaggcat 1921 ttcagtcagt tgctcaatgt acctataacc agaccgttca gctggatatt acggcctttt 1981 taaagaccgt aaagaaaaat aagcacaagt tttatccggc ctttattcac attcttgccc 2041 gcctgatgaa tgctcatccg gaattccgta tggcaatgaa agacggtgag ctggtgatat 2101 gggatagtgt tcacccttgt tacaccgttt tccatgagca aactgaaacg ttttcatcgc 2161 tctggagtga ataccacgac gatttccggc agtttctaca catatattcg caagatgtgg 2221 cgtgttacgg tgaaaacctg gcctatttcc ctaaagggtt tattgagaat atgtttttcg 2281 tctcagccaa tccctgggtg agtttcacca gttttgattt aaacgtggcc aatatggaca 2341 acttcttcgc ccccgttttc accatgggca aatattatac gcaaggcgac aaggtgctga 2401 tgccgctggc gattcaggtt catcatgccg tttgtgatgg cttccatgtc ggcagaatgc 2461 ttaatgaatt acaacagtac tgcgatgagt ggcagggcgg ggcgtaattt ttttaaggca 2521 gttattggtg cccttaaacg cctggttgct acgcctgaat aagtgataat aagcggatga 2581 atggcagaaa ttcgaaagca aattcgaccc ggtcgtcggt tcagggcagg gtcgttaaat 2641 agccgcttat gtctattgct ggtttaccgg tttattgact accggaagca gtgtgaccgt 2701 gtgcttctca aatgcctgag gccagtttgc tcaggctctc cccgtggagg taataattga 2761 cgatatgatc atttattctg cctcccagag cctgataaaa acggttagcg cttcgttaat 2821 acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata 2881 atggtgcagg gcgcttgttt cggcgtgggt atggtggcag gccccgtggc cgggggactg 2941 ttgggcgctg ccggcacctg tcctacgagt tgcatgataa agaagacagt cataagtgcg 3001 gcgacgatag tcatgccccg cgcccaccgg aaggagctac cggcagcggt gcggactgtt 3061 gtaactcaga ataagaaatg aggccgctca tggcgttgac tctcagtcat agtatcgtgg 3121 tatcaccggt tggttccact ctctgttgcg ggcaacttca gcagcacgta ggggacttcc 3181 gcgtttccag actttacgaa acacggaaac cgaagaccat tcatgttgtt gctcaggtcg 3241 cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct 3301 aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc acgatcatgc 3361 gcacccgtgg ccaggaccca acgctgcccg agatgcgccg cgtgcggctg ctggagatgg 3421 cggacgcgat ggatatgttc tgccaagggt tggtttgcgc attcacagtt ctccgcaaga 3481 attgattggc tccaattctt ggagtggtga atccgttagc gaggtgccgc cggcttccat 3541 tcaggtcgag gtggcccggc tccatgcacc gcgacgcaac gcggggaggc agacaaggta 3601 tagggcggcg cctacaatcc atgccaaccc gttccatgtg ctcgccgagg cggcataaat 3661 cgccgtgacg atcagcggtc caatgatcga agttaggctg gtaagagccg cgagcgatcc 3721 ttgaagctgt ccctgatggt cgtcatctac ctgcctggac agcatggcct gcaacgcggg 3781 catcccgatg ccgccggaag cgagaagaat cataatgggg aaggccatcc agcctcgcgt 3841 cgcgaacgcc agcaagacgt agcccagcgc gtcggccgcc atgccggcga taatggcctg 3901 cttctcgccg aaacgtttgg tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa 3961 gattccgaat accgcaagcg acaggccgat catcgtcgcg ctccagcgaa agcggtcctc 4021 gccgaaaatg acccagagcg ctgccggcac ctgtcctacg agttgcatga taaagaagac 4081 agtcataagt gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt 4141 gaaggctctc aagggcatcg gtcgacgctc tcccttatgc gactcctgca ttaggaagca 4201 gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga 4261 gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc 4321 gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc 4381 gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat 4441 ccacaggacg ggtgtggtcg ccatgatcgc gtagtcgata gtggctccaa gtagcgaagc 4501 gagcaggact gggcggcggc caaagcggtc ggacagtgct ccgagaacgg gtgcgcatag 4561 aaattgcatc aacgcatata gcgctagcag cacgccatag tgactggcga tgctgtcgga 4621 atggacgatc acactactct tcttttgcct attataacat gaaaacgctt taacagaaag 4681 aataggaagg tatccgactg ctggtattaa ccctctttct caagttatca tcggcaatgt 4741 tgaatggagt ccattcaaaa cagcatagct ctaaaacatg gaactcaaca agtctcagtg 4801 tgctgaagtt ttgggaccat tcaaaacagc atagctctaa aacgagacct ttgagcttcc 4861 gagactggtc tcagttttgg gaccattcaa aacagcatag ctctaaaacc tcgtagacta 4921 tttttgtcta aaaaatttcg taatcgcact atttgtctca gctagacttc agtcttgaaa 4981 agcccctgta ttactgcatt tattaagagt attataccat atttttagtt attaagaaat 5041 aatcttcatc taaaatatac ttcagtcacc tcctagctga ctcaaatcaa tgcgtgtttc 5101 ataaagacca gtgatggatt gatggataag agtggcatct aaaacttctt ttgtagacgt 5161 atatcgttta cgatcaattg ttgtatcaaa atatttaaaa gcagcgggag ctccaagatt 5221 cgtcaacgta aataaatgaa taatattttc tgcttgttca cgtattggtt tgtctctatg 5281 tttgttatat gcactaagaa ctttatctaa attggcatct gctaaaataa cacgcttaga 5341 aaattcactg atttgctcaa taatctcatc taaataatgc ttatgctgct ccacaaacaa 5401 ttgtttttgt tcgttatctt ctggactacc cttcaacttt tcataatgac tagctaaata 5461 taaaaaattc acatatttgc ttggcagagc cagctcattt cctttttgta attctccggc 5521 actagccagc atccgtttac gaccgttttc taactcaaaa agactatatt taggtagttt 5581 aatgattaag tcttttttaa cttccttata tcctttagct tctaaaaagt caatcggatt 5641 tttttcaaag gaacttcttt ccataattgt gatccctagt aactctttaa cggattttaa 5701 cttcttcgat ttcccttttt ccaccttagc aaccactagg actgaataag ctaccgttgg 5761 actatcaaaa ccaccatatt tttttggatc ccagtctttt ttacgagcaa taagcttgtc 5821 cgaatttctt tttggtaaaa ttgactcctt ggagaatccg cctgtctgta cttctgtttt 5881 cttgacaata ttgacttggg gcatggacaa tactttgcgc actgtggcaa aatctcgccc 5941 tttatcccag acaatttctc cagtttcccc attagtttcg attagagggc gtttgcgaat 6001 ctctccattt gcaagtgtaa tttctgtttt gaagaagttc atgatattag agtaaaagaa 6061 atattttgcg gttgctttgc ctatttcttg ctcagactta gcaatcattt tacgaacatc 6121 ataaacttta taatcaccat agacaaactc cgattcaagt tttggatatt tcttaatcaa 6181 agcagttcca acgacggcat ttagatacgc atcatgggca tgatggtaat tgttaatctc 6241 acgtacttta tagaattgga aatcttttcg gaagtcagaa actaatttag attttaaggt 6301 aatcacttta acctctcgaa taagtttatc attttcatcg tatttagtat tcatgcgact 6361 atccaaaatt tgtgccacat gcttagtgat ttggcgagtt tcaaccaatt ggcgtttgat 6421 aaaaccagct ttatcaagtt cactcaaacc tccacgttca gctttcgtta aattatcaaa 6481 cttacgttga gtgattaact tggcgtttag aagttgtctc caatagtttt tcatcttttt 6541 gactacttct tcacttggaa cgttatccga tttaccacga tttttatcag aacgcgttaa 6601 gaccttattg tctattgaat cgtctttaag gaaactttgt ggaacaatgt gatcgacatc 6661 ataatcactt aaacgattaa tatctaattc ttggtccaca tacatgtctc ttccattttg 6721 gagataatag agatagagct tttcattttg caattgagta ttttcaacag gatgctcttt 6781 aagaatctga cttcctaatt ctttgatacc ttcttcgatt cgtttcatac gctctcgcga 6841 atttttctgg cccttttgag ttgtctgatt ttcacgtgcc atttcaataa cgatattttc 6901 tggcttatgc cgccccatta ctttgaccaa ttcatcaaca acttttacag tctgtaaaat 6961 acctttttta atagcagggc taccagctaa atttgcaata tgttcatgta aactatcgcc 7021 ttgtccagac acttgtgctt tttgaatgtc ttctttaaat gtcaaactat catcatggat 7081 cagctgcata aaattgcgat tggcaaaacc atctgatttc aaaaaatcta atattgtttt 7141 gccagattgc ttatccctaa taccattaat caattttcga gacaaacgtc cccaaccagt 7201 ataacggcga cgtttaagct gtttcatcac cttatcatca aagaggtgag catatgtttt 7261 aagtctttcc tcaatcatct ccctatcttc aaataaggtc aatgttaaaa caatatcctc 7321 taagatatct tcattttctt cattatccaa aaaatcttta tctttaataa tttttagcaa 7381 atcatggtag gtacctaatg aagcattaaa tctatcttca actcctgaaa tttcaacact 7441 atcaaaacat tctatttttt tgaaataatc ttcttttaat tgcttaacgg ttacttttcg 7501 atttgttttg aagagtaaat caacaatggc tttcttctgt tcacctgaaa gaaatgctgg 7561 ttttcgcatt ccttcagtaa catatttgac ctttgtcaat tcgttataaa ccgtaaaata 7621 ctcataaagc aaactatgtt ttggtagtac tttttcattt ggaagatttt tatcaaagtt 7681 tgtcatgcgt tcaataaatg attgagctga agcaccttta tcgacaactt cttcaaaatt 7741 ccatggggta attgtttctt cagacttccg agtcatccat gcaaaacgac tattgccacg 7801 cgccaatgga ccaacataat aaggaattcg aaaagtcaag attttttcaa tcttctcacg 7861 attgtctttt aaaaatggat aaaagtcttc ttgtcttctc aaaatagcat gcagctcacc 7921 caagtgaatt tgatggggaa tagagccgtt gtcaaaggtc cgttgcttgc gcagcaaatc 7981 ttcacgattt agtttcacca ataattcctc agtaccatcc attttttcta aaattggttt 8041 gataaattta taaaattctt cttggctagc tcccccatca atataacctg catatccgtt 8101 ttttgattga tcaaaaaaga tttctttata cttttctgga agttgttgtc gaactaaagc 8161 ttttaaaaga gtcaagtctt gatgatgttc atcgtagcgt ttaatcattg aagctgatag 8221 gggagcctta gttatttcag tatttactct taggatatct gaaagtaaaa tagcatctga 8281 taaattctta gctgccaaaa acaaatcagc atattgatct ccaatttgcg ccaataaatt 8341 atctaaatca tcatcgtaag tatcttttga aagctgtaat ttagcatctt ctgccaaatc 8401 aaaatttgat ttaaaattag gggtcaaacc caatgacaaa gcaatgagat tcccaaataa 8461 gccatttttc ttctcaccgg ggagctgagc aatgagattt tctaatcgtc ttgatttact 8521 caatcgtgca gaaagaatcg ctttagcatc tactccactt gcgttaatag ggttttcttc 8581 aaataattga ttgtaggttt gtaccaactg gataaatagt ttgtccacat cactattatc 8641 aggatttaaa tctccctcaa tcaaaaaatg accacgaaac ttaatcatat gcgctaaggc 8701 caaatagatt aagcgcaaat ccgctttatc agtagaatct accaattttt ttcgcagatg 8761 atagatagtt ggatatttct catgataagc aacttcatct actatatttc caaaaatagg 8821 atgacgttca tgcttcttgt cttcttccac caaaaaagac tcttcaagtc gatgaaagaa 8881 actatcatct actttcgcca tctcatttga aaaaatctcc tgtagataac aaatacgatt 8941 cttccgacgt gtataccttc tacgagctgt ccgtttgaga cgagtcgctt ccgctgtctc 9001 tccactgtca aataaaagag cccctataag attttttttg atactgtggc ggtctgtatt 9061 tcccagaacc ttgaactttt tagacggaac cttatattca tcagtgatca ccgcccatcc 9121 gacgctattt gtgccgatat ctaagcctat tgagtatttc ttatccattt ttgcctccta 9181 aaataaaaag tttaaattaa atccataatg agtttgatga tttcaataat agttttaatg 9241 acctccgaaa ttagtttaat atgctttaat ttttcttttt caaaatatct cttcaaaaaa 9301 tattacccaa tacttaataa taaata //