LOCUS Exported 8505 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Cas9n (D10A nickase mutant) from S. pyogenes. ACCESSION . VERSION . KEYWORDS pSpCas9n(BB) (PX460) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8505) AUTHORS Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F TITLE Genome engineering using the CRISPR-Cas9 system. JOURNAL Nat Protoc. 2013 Nov;8(11):2281-308. doi: 10.1038/nprot.2013.143. Epub 2013 Oct 24. PUBMED 24157548 REFERENCE 2 (bases 1 to 8505) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8505 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 1..241 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 1..21 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 172..191 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 268..343 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" enhancer 440..725 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer; contains an 18-bp deletion relative to the standard CMV enhancer" promoter 727..1004 /label=chicken beta-actin promoter intron 1005..1232 /label=hybrid intron /note="hybrid between chicken beta-actin (CBA) and minute virus of mice (MMV) introns (Gray et al., 2011)" regulatory 1244..1253 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1253..1318 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 1325..1345 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 1370..5470 /codon_start=1 /product="nickase mutant of the Cas9 endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /label=Cas9(D10A) /note="generates RNA-guided single strand nicks in DNA due to the D10A mutation in the RuvC catalytic domain" /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGD" CDS 5471..5518 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" primer_bind complement(5546..5563) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 5552..5759 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" repeat_region 5768..5908 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 5768..5897 /label=AAV2 ITR rep_origin 5983..6438 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(6070..6089) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 6280..6301 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(6455..6474) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 6574..6596 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(6634..6652) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 6720..6824 /gene="bla" /label=AmpR promoter CDS 6825..7685 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(7043..7062) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 7856..8444 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 8345..8364 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 241 cgaaacaccg ggtcttcgag aagacctgtt ttagagctag aaatagcaag ttaaaataag 301 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg ttttagagct 361 agaaatagca agttaaaata aggctagtcc gtttttagcg cgtgcgccaa ttctgcagac 421 aaatggctct agaggtaccc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 481 ccaacgaccc ccgcccattg acgtcaatag taacgccaat agggactttc cattgacgtc 541 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc 601 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tgtgcccagt 661 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta 721 ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc ccctccccac 781 ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg gcgggggggg 841 ggggggggcg cgcgccaggc ggggcggggc ggggcgrggg gcggggcggg gcgaggcgga 901 gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa gtttcctttt atggcgaggc 961 ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgcgct 1021 gccttcgccc cgtgccccgc tccgccgccg cctcgcgccg cccgccccgg ctctgactga 1081 ccgcgttact cccacaggtg agcgggcggg acggcccttc tcctccgggc tgtaattagc 1141 tgagcaagag gtaagggttt aagggatggt tggttggtgg ggtattaatg tttaattacc 1201 tggagcacct gcctgaaatc actttttttc aggttggacc ggtgccacca tggactataa 1261 ggaccacgac ggagactaca aggatcatga tattgattac aaagacgatg acgataagat 1321 ggccccaaag aagaagcgga aggtcggtat ccacggagtc ccagcagccg acaagaagta 1381 cagcatcggc ctggccatcg gcaccaactc tgtgggctgg gccgtgatca ccgacgagta 1441 caaggtgccc agcaagaaat tcaaggtgct gggcaacacc gaccggcaca gcatcaagaa 1501 gaacctgatc ggagccctgc tgttcgacag cggcgaaaca gccgaggcca cccggctgaa 1561 gagaaccgcc agaagaagat acaccagacg gaagaaccgg atctgctatc tgcaagagat 1621 cttcagcaac gagatggcca aggtggacga cagcttcttc cacagactgg aagagtcctt 1681 cctggtggaa gaggataaga agcacgagcg gcaccccatc ttcggcaaca tcgtggacga 1741 ggtggcctac cacgagaagt accccaccat ctaccacctg agaaagaaac tggtggacag 1801 caccgacaag gccgacctgc ggctgatcta tctggccctg gcccacatga tcaagttccg 1861 gggccacttc ctgatcgagg gcgacctgaa ccccgacaac agcgacgtgg acaagctgtt 1921 catccagctg gtgcagacct acaaccagct gttcgaggaa aaccccatca acgccagcgg 1981 cgtggacgcc aaggccatcc tgtctgccag actgagcaag agcagacggc tggaaaatct 2041 gatcgcccag ctgcccggcg agaagaagaa tggcctgttc ggcaacctga ttgccctgag 2101 cctgggcctg acccccaact tcaagagcaa cttcgacctg gccgaggatg ccaaactgca 2161 gctgagcaag gacacctacg acgacgacct ggacaacctg ctggcccaga tcggcgacca 2221 gtacgccgac ctgtttctgg ccgccaagaa cctgtccgac gccatcctgc tgagcgacat 2281 cctgagagtg aacaccgaga tcaccaaggc ccccctgagc gcctctatga tcaagagata 2341 cgacgagcac caccaggacc tgaccctgct gaaagctctc gtgcggcagc agctgcctga 2401 gaagtacaaa gagattttct tcgaccagag caagaacggc tacgccggct acattgacgg 2461 cggagccagc caggaagagt tctacaagtt catcaagccc atcctggaaa agatggacgg 2521 caccgaggaa ctgctcgtga agctgaacag agaggacctg ctgcggaagc agcggacctt 2581 cgacaacggc agcatccccc accagatcca cctgggagag ctgcacgcca ttctgcggcg 2641 gcaggaagat ttttacccat tcctgaagga caaccgggaa aagatcgaga agatcctgac 2701 cttccgcatc ccctactacg tgggccctct ggccagggga aacagcagat tcgcctggat 2761 gaccagaaag agcgaggaaa ccatcacccc ctggaacttc gaggaagtgg tggacaaggg 2821 cgcttccgcc cagagcttca tcgagcggat gaccaacttc gataagaacc tgcccaacga 2881 gaaggtgctg cccaagcaca gcctgctgta cgagtacttc accgtgtata acgagctgac 2941 caaagtgaaa tacgtgaccg agggaatgag aaagcccgcc ttcctgagcg gcgagcagaa 3001 aaaggccatc gtggacctgc tgttcaagac caaccggaaa gtgaccgtga agcagctgaa 3061 agaggactac ttcaagaaaa tcgagtgctt cgactccgtg gaaatctccg gcgtggaaga 3121 tcggttcaac gcctccctgg gcacatacca cgatctgctg aaaattatca aggacaagga 3181 cttcctggac aatgaggaaa acgaggacat tctggaagat atcgtgctga ccctgacact 3241 gtttgaggac agagagatga tcgaggaacg gctgaaaacc tatgcccacc tgttcgacga 3301 caaagtgatg aagcagctga agcggcggag atacaccggc tggggcaggc tgagccggaa 3361 gctgatcaac ggcatccggg acaagcagtc cggcaagaca atcctggatt tcctgaagtc 3421 cgacggcttc gccaacagaa acttcatgca gctgatccac gacgacagcc tgacctttaa 3481 agaggacatc cagaaagccc aggtgtccgg ccagggcgat agcctgcacg agcacattgc 3541 caatctggcc ggcagccccg ccattaagaa gggcatcctg cagacagtga aggtggtgga 3601 cgagctcgtg aaagtgatgg gccggcacaa gcccgagaac atcgtgatcg aaatggccag 3661 agagaaccag accacccaga agggacagaa gaacagccgc gagagaatga agcggatcga 3721 agagggcatc aaagagctgg gcagccagat cctgaaagaa caccccgtgg aaaacaccca 3781 gctgcagaac gagaagctgt acctgtacta cctgcagaat gggcgggata tgtacgtgga 3841 ccaggaactg gacatcaacc ggctgtccga ctacgatgtg gaccatatcg tgcctcagag 3901 ctttctgaag gacgactcca tcgacaacaa ggtgctgacc agaagcgaca agaaccgggg 3961 caagagcgac aacgtgccct ccgaagaggt cgtgaagaag atgaagaact actggcggca 4021 gctgctgaac gccaagctga ttacccagag aaagttcgac aatctgacca aggccgagag 4081 aggcggcctg agcgaactgg ataaggccgg cttcatcaag agacagctgg tggaaacccg 4141 gcagatcaca aagcacgtgg cacagatcct ggactcccgg atgaacacta agtacgacga 4201 gaatgacaag ctgatccggg aagtgaaagt gatcaccctg aagtccaagc tggtgtccga 4261 tttccggaag gatttccagt tttacaaagt gcgcgagatc aacaactacc accacgccca 4321 cgacgcctac ctgaacgccg tcgtgggaac cgccctgatc aaaaagtacc ctaagctgga 4381 aagcgagttc gtgtacggcg actacaaggt gtacgacgtg cggaagatga tcgccaagag 4441 cgagcaggaa atcggcaagg ctaccgccaa gtacttcttc tacagcaaca tcatgaactt 4501 tttcaagacc gagattaccc tggccaacgg cgagatccgg aagcggcctc tgatcgagac 4561 aaacggcgaa accggggaga tcgtgtggga taagggccgg gattttgcca ccgtgcggaa 4621 agtgctgagc atgccccaag tgaatatcgt gaaaaagacc gaggtgcaga caggcggctt 4681 cagcaaagag tctatcctgc ccaagaggaa cagcgataag ctgatcgcca gaaagaagga 4741 ctgggaccct aagaagtacg gcggcttcga cagccccacc gtggcctatt ctgtgctggt 4801 ggtggccaaa gtggaaaagg gcaagtccaa gaaactgaag agtgtgaaag agctgctggg 4861 gatcaccatc atggaaagaa gcagcttcga gaagaatccc atcgactttc tggaagccaa 4921 gggctacaaa gaagtgaaaa aggacctgat catcaagctg cctaagtact ccctgttcga 4981 gctggaaaac ggccggaaga gaatgctggc ctctgccggc gaactgcaga agggaaacga 5041 actggccctg ccctccaaat atgtgaactt cctgtacctg gccagccact atgagaagct 5101 gaagggctcc cccgaggata atgagcagaa acagctgttt gtggaacagc acaagcacta 5161 cctggacgag atcatcgagc agatcagcga gttctccaag agagtgatcc tggccgacgc 5221 taatctggac aaagtgctgt ccgcctacaa caagcaccgg gataagccca tcagagagca 5281 ggccgagaat atcatccacc tgtttaccct gaccaatctg ggagcccctg ccgccttcaa 5341 gtactttgac accaccatcg accggaagag gtacaccagc accaaagagg tgctggacgc 5401 caccctgatc caccagagca tcaccggcct gtacgagaca cggatcgacc tgtctcagct 5461 gggaggcgac aaaaggccgg cggccacgaa aaaggccggc caggcaaaaa agaaaaagta 5521 agaattccta gagctcgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt 5581 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc 5641 taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt 5701 ggggtggggc aggacagcaa gggggaggat tgggaagaga atagcaggca tgctggggag 5761 cggccgcagg aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc 5821 actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg 5881 agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc ggtattttct ccttacgcat 5941 ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc tgtagcggcg 6001 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgcct 6061 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc 6121 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg 6181 accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg 6241 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg 6301 gaacaacact caactctatc tcgggctatt cttttgattt ataagggatt ttgccgattt 6361 cggtctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa 6421 tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat gccgcatagt 6481 taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc 6541 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt 6601 caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg 6661 ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc 6721 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac 6781 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt 6841 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag 6901 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg 6961 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa 7021 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc 7081 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag 7141 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa 7201 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc 7261 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg 7321 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa 7381 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa 7441 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg 7501 gctggtttat tgctgataaa tctggagccg gtgagcgtgg aagccgcggt atcattgcag 7561 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg 7621 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt 7681 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt 7741 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac 7801 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag 7861 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg 7921 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca 7981 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga 8041 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca 8101 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc 8161 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca 8221 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa 8281 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc 8341 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc 8401 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg 8461 cctttttacg gttcctggcc ttttgctggc cttttgctca catgt //