LOCUS Exported 6182 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Plasmid constitutively expressing dCas9 protein. ACCESSION . VERSION . KEYWORDS pdCas9-J23109 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6182) AUTHORS Guo J, Wang T, Guan C, Liu B, Luo C, Xie Z, Zhang C, Xing XH TITLE Improved sgRNA design in bacteria via genome-wide activity profiling. JOURNAL Nucleic Acids Res. 2018 Aug 21;46(14):7052-7069. doi: 10.1093/nar/gky572. PUBMED 29982721 REFERENCE 2 (bases 1 to 6182) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6182 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 1..4107 /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" terminator 4131..4202 /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" terminator 4218..4245 /label=T7Te terminator /note="phage T7 early transcription terminator" primer_bind complement(4273..4290) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4407..4952) /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(4487..4506) /label=pBR322ori-F /note="pBR322 origin, forward primer" terminator 5066..5160 /label=lambda t0 terminator /note="transcription terminator from phage lambda" CDS complement(5184..5999) /codon_start=1 /gene="aph(3')-Ia" /product="aminoglycoside phosphotransferase" /label=KanR /note="confers resistance to kanamycin in bacteria or G418 (Geneticin(R)) in eukaryotes" /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF" primer_bind 5907..5926 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 5991..6010 /label=pENTR-R /note="pENTR vectors, reverse primer" RBS 6165..6176 /note="strong bacterial ribosome binding site (Elowitz and Leibler, 2000)" ORIGIN 1 atggataaga aatactcaat aggcttagct atcggcacaa atagcgtcgg atgggcggtg 61 atcactgatg aatataaggt tccgtctaaa aagttcaagg ttctgggaaa tacagaccgc 121 cacagtatca aaaaaaatct tataggggct cttttatttg acagtggaga gacagcggaa 181 gcgactcgtc tcaaacggac agctcgtaga aggtatacac gtcggaagaa tcgtatttgt 241 tatctacagg agattttttc aaatgagatg gcgaaagtag atgatagttt ctttcatcga 301 cttgaagagt cttttttggt ggaagaagac aagaagcatg aacgtcatcc tatttttgga 361 aatatagtag atgaagttgc ttatcatgag aaatatccaa ctatctatca tctgcgaaaa 421 aaattggtag attctactga taaagcggat ttgcgcttaa tctatttggc cttagcgcat 481 atgattaagt ttcgtggtca ttttttgatt gagggagatt taaatcctga taatagtgat 541 gtggacaaac tatttatcca gttggtacaa acctacaatc aattatttga agaaaaccct 601 attaacgcaa gtggagtaga tgctaaagcg attctttctg cacgattgag taaatcaaga 661 cgattagaaa atctcattgc tcagctcccc ggtgagaaga aaaatggctt atttgggaat 721 ctcattgctt tgtcattggg tttgacccct aattttaaat caaattttga tttggcagaa 781 gatgctaaat tacagctttc aaaagatact tacgatgatg atttagataa tttattggcg 841 caaattggag atcaatatgc tgatttgttt ttggcagcta agaatttatc agatgctatt 901 ttactttcag atatcctaag agtaaatact gaaataacta aggctcccct atcagcttca 961 atgattaaac gctacgatga acatcatcaa gacttgactc ttttaaaagc tttagttcga 1021 caacaacttc cagaaaagta taaagaaatc ttttttgatc aatcaaaaaa cggatatgca 1081 ggttatattg atgggggagc tagccaagaa gaattttata aatttatcaa accaatttta 1141 gaaaaaatgg atggtactga ggaattattg gtgaaactaa atcgtgaaga tttgctgcgc 1201 aagcaacgga cctttgacaa cggctctatt ccccatcaaa ttcacttggg tgagctgcat 1261 gctattttga gaagacaaga agacttttat ccatttttaa aagacaatcg tgagaagatt 1321 gaaaaaatct tgacttttcg aattccttat tatgttggtc cattggcgcg tggcaatagt 1381 cgttttgcat ggatgactcg gaagtctgaa gaaacaatta ccccatggaa ttttgaagaa 1441 gttgtcgata aaggtgcttc agctcaatca tttattgaac gcatgacaaa ctttgataaa 1501 aatcttccaa atgaaaaagt actaccaaaa catagtttgc tttatgagta ttttacggtt 1561 tataacgaat tgacaaaggt caaatatgtt actgaaggaa tgcgaaaacc agcatttctt 1621 tcaggtgaac agaagaaagc cattgttgat ttactcttca aaacaaatcg aaaagtaacc 1681 gttaagcaat taaaagaaga ttatttcaaa aaaatagaat gttttgatag tgttgaaatt 1741 tcaggagttg aagatagatt taatgcttca ttaggtacct accatgattt gctaaaaatt 1801 attaaagata aagatttttt ggataatgaa gaaaatgaag atatcttaga ggatattgtt 1861 ttaacattga ccttatttga agatagggag atgattgagg aaagacttaa aacatatgct 1921 cacctctttg atgataaggt gatgaaacag cttaaacgtc gccgttatac tggttgggga 1981 cgtttgtctc gaaaattgat taatggtatt agggataagc aatctggcaa aacaatatta 2041 gattttttga aatcagatgg ttttgccaat cgcaatttta tgcagctgat ccatgatgat 2101 agtttgacat ttaaagaaga cattcaaaaa gcacaagtgt ctggacaagg cgatagttta 2161 catgaacata ttgcaaattt agctggtagc cctgctatta aaaaaggtat tttacagact 2221 gtaaaagttg ttgatgaatt ggtcaaagta atggggcggc ataagccaga aaatatcgtt 2281 attgaaatgg cacgtgaaaa tcagacaact caaaagggcc agaaaaattc gcgagagcgt 2341 atgaaacgaa tcgaagaagg tatcaaagaa ttaggaagtc agattcttaa agagcatcct 2401 gttgaaaata ctcaattgca aaatgaaaag ctctatctct attatctcca aaatggaaga 2461 gacatgtatg tggaccaaga attagatatt aatcgtttaa gtgattatga tgtcgatgcc 2521 attgttccac aaagtttcct taaagacgat tcaatagaca ataaggtctt aacgcgttct 2581 gataaaaatc gtggtaaatc ggataacgtt ccaagtgaag aagtagtcaa aaagatgaaa 2641 aactattgga gacaacttct aaacgccaag ttaatcactc aacgtaagtt tgataattta 2701 acgaaagctg aacgtggagg tttgagtgaa cttgataaag ctggttttat caaacgccaa 2761 ttggttgaaa ctcgccaaat cactaagcat gtggcacaaa ttttggatag tcgcatgaat 2821 actaaatacg atgaaaatga taaacttatt cgagaggtta aagtgattac cttaaaatct 2881 aaattagttt ctgacttccg aaaagatttc caattctata aagtacgtga gattaacaat 2941 taccatcatg cccatgatgc gtatctaaat gccgtcgttg gaactgcttt gattaagaaa 3001 tatccaaaac ttgaatcgga gtttgtctat ggtgattata aagtttatga tgttcgtaaa 3061 atgattgcta agtctgagca agaaataggc aaagcaaccg caaaatattt cttttactct 3121 aatatcatga acttcttcaa aacagaaatt acacttgcaa atggagagat tcgcaaacgc 3181 cctctaatcg aaactaatgg ggaaactgga gaaattgtct gggataaagg gcgagatttt 3241 gccacagtgc gcaaagtatt gtccatgccc caagtcaata ttgtcaagaa aacagaagta 3301 cagacaggcg gattctccaa ggagtcaatt ttaccaaaaa gaaattcgga caagcttatt 3361 gctcgtaaaa aagactggga tccaaaaaaa tatggtggtt ttgatagtcc aacggtagct 3421 tattcagtcc tagtggttgc taaggtggaa aaagggaaat cgaagaagtt aaaatccgtt 3481 aaagagttac tagggatcac aattatggaa agaagttcct ttgaaaaaaa tccgattgac 3541 tttttagaag ctaaaggata taaggaagtt aaaaaagact taatcattaa actacctaaa 3601 tatagtcttt ttgagttaga aaacggtcgt aaacggatgc tggctagtgc cggagaatta 3661 caaaaaggaa atgagctggc tctgccaagc aaatatgtga attttttata tttagctagt 3721 cattatgaaa agttgaaggg tagtccagaa gataacgaac aaaaacaatt gtttgtggag 3781 cagcataagc attatttaga tgagattatt gagcaaatca gtgaattttc taagcgtgtt 3841 attttagcag atgccaattt agataaagtt cttagtgcat ataacaaaca tagagacaaa 3901 ccaatacgtg aacaagcaga aaatattatt catttattta cgttgacgaa tcttggagct 3961 cccgctgctt ttaaatattt tgatacaaca attgatcgta aacgatatac gtctacaaaa 4021 gaagttttag atgccactct tatccatcaa tccatcactg gtctttatga aacacgcatt 4081 gatttgagtc agctaggagg tgactaactc gagtaaggat ctccaggcat caaataaaac 4141 gaaaggctca gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg gtgaacgctc 4201 tctactagag tcacactggc tcaccttcgg gtgggccttt ctgcgtttat acctagggat 4261 atattccgct tcctcgctca ctgactcgct acgctcggtc gttcgactgc ggcgagcgga 4321 aatggcttac gaacggggcg gagatttcct ggaagatgcc aggaagatac ttaacaggga 4381 agtgagaggg ccgcggcaaa gccgtttttc cataggctcc gcccccctga caagcatcac 4441 gaaatctgac gctcaaatca gtggtggcga aacccgacag gactataaag ataccaggcg 4501 tttccccctg gcggctccct cgtgcgctct cctgttcctg cctttcggtt taccggtgtc 4561 attccgctgt tatggccgcg tttgtctcat tccacgcctg acactcagtt ccgggtaggc 4621 agttcgctcc aagctggact gtatgcacga accccccgtt cagtccgacc gctgcgcctt 4681 atccggtaac tatcgtcttg agtccaaccc ggaaagacat gcaaaagcac cactggcagc 4741 agccactggt aattgattta gaggagttag tcttgaagtc atgcgccggt taaggctaaa 4801 ctgaaaggac aagttttggt gactgcgctc ctccaagcca gttacctcgg ttcaaagagt 4861 tggtagctca gagaaccttc gaaaaaccgc cctgcaaggc ggttttttcg ttttcagagc 4921 aagagattac gcgcagacca aaacgatctc aagaagatca tcttattaat cagataaaat 4981 atttctagat ttcagtgcaa tttatctctt caaatgtagc acctgaagtc agccccatac 5041 gatataagtt gttactagtg cttggattct caccaataaa aaacgcccgg cggcaaccga 5101 gcgttctgaa caaatccaga tggagttctg aggtcattac tggatctatc aacaggagtc 5161 caagcgagct cgatatcaaa ttattagaaa aactcatcga gcatcaaatg aaactgcaat 5221 ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga 5281 gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg 5341 actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt 5401 gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagctt atgcatttct 5461 ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc 5521 aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa 5581 ggacaattac aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca 5641 atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt cccggggatc 5701 gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga 5761 ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg 5821 ctacctttgc catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcgatag 5881 attgtcgcac ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca 5941 tccatgttgg aatttaatcg cggcctcgag caagacgttt cccgttgaat atggctcata 6001 acaccccttg tattactgtt tatgtaagca gacagtttta ttgttcatga tgatatattt 6061 ttatcttgtg caatgtaaca tcagagattt tgagacacaa cgtggctttc cctgcagttt 6121 acagctagct cagtcctagg gactgtgcta gcggccgcag atctaaagag gagaaaggat 6181 ct //