LOCUS Exported 4272 bp ds-DNA linear SYN 17-MAY-2021 DEFINITION Expresses high specificity SpCas9. Px330-like plasmid.. ACCESSION . VERSION . KEYWORDS eSpCas9(1.1) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4272) AUTHORS Slaymaker IM, Gao L, Zetsche B, Scott DA, Yan WX, Zhang F TITLE Rationally engineered Cas9 nucleases with improved specificity. JOURNAL Science. 2015 Dec 1. pii: aad5227. PUBMED 26628643 REFERENCE 2 (bases 1 to 4272) AUTHORS . TITLE Direct Submission JOURNAL Exported May 17, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4272 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 4..69 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 76..96 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 121..4221 /codon_start=1 /product="Cas9 endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system, mutated to improve targeting specificity (Slaymaker et al., 2016)" /label=eSpCas9(1.1) /note="carries the mutations K848A, K1003A, and R1060A" /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLADDSIDNKVLTRSDKNRGKSDNVPS EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPALESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE ITLANGEIRKAPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGD" CDS 4222..4269 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" ORIGIN 1 atggactata aggaccacga cggagactac aaggatcatg atattgatta caaagacgat 61 gacgataaga tggccccaaa gaagaagcgg aaggtcggta tccacggagt cccagcagcc 121 gacaagaagt acagcatcgg cctggacatc ggcaccaact ctgtgggctg ggccgtgatc 181 accgacgagt acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac 241 agcatcaaga agaacctgat cggagccctg ctgttcgaca gcggcgaaac agccgaggcc 301 acccggctga agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat 361 ctgcaagaga tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg 421 gaagagtcct tcctggtgga agaggataag aagcacgagc ggcaccccat cttcggcaac 481 atcgtggacg aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa 541 ctggtggaca gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg 601 atcaagttcc ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg 661 gacaagctgt tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc 721 aacgccagcg gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagacgg 781 ctggaaaatc tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggaaacctg 841 attgccctga gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat 901 gccaaactgc agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag 961 atcggcgacc agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg 1021 ctgagcgaca tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg 1081 atcaagagat acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag 1141 cagctgcctg agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc 1201 tacattgacg gcggagccag ccaggaagag ttctacaagt tcatcaagcc catcctggaa 1261 aagatggacg gcaccgagga actgctcgtg aagctgaaca gagaggacct gctgcggaag 1321 cagcggacct tcgacaacgg cagcatcccc caccagatcc acctgggaga gctgcacgcc 1381 attctgcggc ggcaggaaga tttttaccca ttcctgaagg acaaccggga aaagatcgag 1441 aagatcctga ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga 1501 ttcgcctgga tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg 1561 gtggacaagg gcgcttccgc ccagagcttc atcgagcgga tgaccaactt cgataagaac 1621 ctgcccaacg agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtat 1681 aacgagctga ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc cttcctgagc 1741 ggcgagcaga aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg 1801 aagcagctga aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc 1861 ggcgtggaag atcggttcaa cgcctccctg ggcacatacc acgatctgct gaaaattatc 1921 aaggacaagg acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg 1981 accctgacac tgtttgagga cagagagatg atcgaggaac ggctgaaaac ctatgcccac 2041 ctgttcgacg acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcagg 2101 ctgagccgga agctgatcaa cggcatccgg gacaagcagt ccggcaagac aatcctggat 2161 ttcctgaagt ccgacggctt cgccaacaga aacttcatgc agctgatcca cgacgacagc 2221 ctgaccttta aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcac 2281 gagcacattg ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg 2341 aaggtggtgg acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc 2401 gaaatggcca gagagaacca gaccacccag aagggacaga agaacagccg cgagagaatg 2461 aagcggatcg aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg 2521 gaaaacaccc agctgcagaa cgagaagctg tacctgtact acctgcagaa tgggcgggat 2581 atgtacgtgg accaggaact ggacatcaac cggctgtccg actacgatgt ggaccatatc 2641 gtgcctcaga gctttctggc cgacgactcc atcgacaaca aggtgctgac cagaagcgac 2701 aagaaccggg gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac 2761 tactggcggc agctgctgaa cgccaagctg attacccaga gaaagttcga caatctgacc 2821 aaggccgaga gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg 2881 gtggaaaccc ggcagatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact 2941 aagtacgacg agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag 3001 ctggtgtccg atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac 3061 caccacgccc acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac 3121 cctgcgctgg aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg 3181 atcgccaaga gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac 3241 atcatgaact ttttcaagac cgagattacc ctggccaacg gcgagatccg gaaggcgcct 3301 ctgatcgaga caaacggcga aaccggggag atcgtgtggg ataagggccg ggattttgcc 3361 accgtgcgga aagtgctgag catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag 3421 acaggcggct tcagcaaaga gtctatcctg cccaagagga acagcgataa gctgatcgcc 3481 agaaagaagg actgggaccc taagaagtac ggcggcttcg acagccccac cgtggcctat 3541 tctgtgctgg tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa 3601 gagctgctgg ggatcaccat catggaaaga agcagcttcg agaagaatcc catcgacttt 3661 ctggaagcca agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac 3721 tccctgttcg agctggaaaa cggccggaag agaatgctgg cctctgccgg cgaactgcag 3781 aagggaaacg aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac 3841 tatgagaagc tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag 3901 cacaagcact acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc 3961 ctggccgacg ctaatctgga caaagtgctg tccgcctaca acaagcaccg ggataagccc 4021 atcagagagc aggccgagaa tatcatccac ctgtttaccc tgaccaatct gggagcccct 4081 gccgccttca agtactttga caccaccatc gaccggaaga ggtacaccag caccaaagag 4141 gtgctggacg ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac 4201 ctgtctcagc tgggaggcga caaaaggccg gcggccacga aaaaggccgg ccaggcaaaa 4261 aagaaaaagt aa //