LOCUS Exported 8089 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expresses the M495V/Y515N/K526E/R661Q (evoCas9) SpCas9 variant in mammalian cells. ACCESSION . VERSION . KEYWORDS pX-evoCas9 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8089) AUTHORS Casini A, Olivieri M, Petris G, Montagna C, Reginato G, Maule G, Lorenzin F, Prandi D, Romanel A, Demichelis F, Inga A, Cereseto A TITLE A highly specific SpCas9 variant is identified by in vivo screening in yeast. JOURNAL Nat Biotechnol. 2018 Jan 29. pii: nbt.4066. doi: 10.1038/nbt.4066. PUBMED 29431739 REFERENCE 2 (bases 1 to 8089) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8089 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 1..21 /label=hU6-F /note="Human U6 promoter, forward primer" promoter 311..588 /label=chicken beta-actin promoter intron 589..816 /label=hybrid intron /note="hybrid between chicken beta-actin (CBA) and minute virus of mice (MMV) introns (Gray et al., 2011)" regulatory 828..837 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 837..902 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 909..929 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 954..5054 /codon_start=1 /product="Streptococcus pyogenes Cas9 variant with improved specificity (Casini et al., 2018)" /label=evoCas9 /note="generates RNA-guided double strand breaks in DNA" /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERVTNFDKNLPNEKVLPKHSLLNEYF TVYNELTEVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL KTYAHLFDDKVMKQLKRRRYTGWGQLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGD" CDS 5055..5102 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" primer_bind complement(5130..5147) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 5136..5343 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" repeat_region 5352..5492 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 5352..5481 /label=AAV2 ITR rep_origin 5567..6022 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(5654..5673) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 5864..5885 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(6039..6058) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 6158..6180 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(6218..6236) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 6304..6408 /gene="bla" /label=AmpR promoter CDS 6409..7269 /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(6627..6646) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 7440..8028 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 7929..7948 /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 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattgtgcc 241 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct 301 attaccatgg tcgaggtgag ccccacgttc tgcttcactc tccccatctc ccccccctcc 361 ccacccccaa ttttgtattt atttattttt taattatttt gtgcagcgat gggggcgggg 421 gggggggggg ggcgcgcgcc aggcggggcg gggcggggcg aggggcgggg cggggcgagg 481 cggagaggtg cggcggcagc caatcagagc ggcgcgctcc gaaagtttcc ttttatggcg 541 aggcggcggc ggcggcggcc ctataaaaag cgaagcgcgc ggcgggcggg agtcgctgcg 601 cgctgccttc gccccgtgcc ccgctccgcc gccgcctcgc gccgcccgcc ccggctctga 661 ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc gggctgtaat 721 tagctgagca agaggtaagg gtttaaggga tggttggttg gtggggtatt aatgtttaat 781 tacctggagc acctgcctga aatcactttt tttcaggttg gaccggtgcc accatggact 841 ataaggacca cgacggagac tacaaggatc atgatattga ttacaaagac gatgacgata 901 agatggcccc aaagaagaag cggaaggtcg gtatccacgg agtcccagca gccgacaaga 961 agtacagcat cggcctggac atcggcacca actctgtggg ctgggccgtg atcaccgacg 1021 agtacaaggt gcccagcaag aaattcaagg tgctgggcaa caccgaccgg cacagcatca 1081 agaagaacct gatcggagcc ctgctgttcg acagcggcga aacagccgag gccacccggc 1141 tgaagagaac cgccagaaga agatacacca gacggaagaa ccggatctgc tatctgcaag 1201 agatcttcag caacgagatg gccaaggtgg acgacagctt cttccacaga ctggaagagt 1261 ccttcctggt ggaagaggat aagaagcacg agcggcaccc catcttcggc aacatcgtgg 1321 acgaggtggc ctaccacgag aagtacccca ccatctacca cctgagaaag aaactggtgg 1381 acagcaccga caaggccgac ctgcggctga tctatctggc cctggcccac atgatcaagt 1441 tccggggcca cttcctgatc gagggcgacc tgaaccccga caacagcgac gtggacaagc 1501 tgttcatcca gctggtgcag acctacaacc agctgttcga ggaaaacccc atcaacgcca 1561 gcggcgtgga cgccaaggcc atcctgtctg ccagactgag caagagcaga cggctggaaa 1621 atctgatcgc ccagctgccc ggcgagaaga agaatggcct gttcggaaac ctgattgccc 1681 tgagcctggg cctgaccccc aacttcaaga gcaacttcga cctggccgag gatgccaaac 1741 tgcagctgag caaggacacc tacgacgacg acctggacaa cctgctggcc cagatcggcg 1801 accagtacgc cgacctgttt ctggccgcca agaacctgtc cgacgccatc ctgctgagcg 1861 acatcctgag agtgaacacc gagatcacca aggcccccct gagcgcctct atgatcaaga 1921 gatacgacga gcaccaccag gacctgaccc tgctgaaagc tctcgtgcgg cagcagctgc 1981 ctgagaagta caaagagatt ttcttcgacc agagcaagaa cggctacgcc ggctacattg 2041 acggcggagc cagccaggaa gagttctaca agttcatcaa gcccatcctg gaaaagatgg 2101 acggcaccga ggaactgctc gtgaagctga acagagagga cctgctgcgg aagcagcgga 2161 ccttcgacaa cggcagcatc ccccaccaga tccacctggg agagctgcac gccattctgc 2221 ggcggcagga agatttttac ccattcctga aggacaaccg ggaaaagatc gagaagatcc 2281 tgaccttccg catcccctac tacgtgggcc ctctggccag gggaaacagc agattcgcct 2341 ggatgaccag aaagagcgag gaaaccatca ccccctggaa cttcgaggaa gtggtggaca 2401 agggcgcttc cgcccagagc ttcatcgagc gggtgaccaa cttcgataag aacctgccca 2461 acgagaaggt gctgcccaag cacagcctgc tgaacgagta cttcaccgtg tataacgagc 2521 tgaccgaagt gaaatacgtg accgagggaa tgagaaagcc cgccttcctg agcggcgagc 2581 agaaaaaggc catcgtggac ctgctgttca agaccaaccg gaaagtgacc gtgaagcagc 2641 tgaaagagga ctacttcaag aaaatcgagt gcttcgactc cgtggaaatc tccggcgtgg 2701 aagatcggtt caacgcctcc ctgggcacat accacgatct gctgaaaatt atcaaggaca 2761 aggacttcct ggacaatgag gaaaacgagg acattctgga agatatcgtg ctgaccctga 2821 cactgtttga ggacagagag atgatcgagg aacggctgaa aacctatgcc cacctgttcg 2881 acgacaaagt gatgaagcag ctgaagcggc ggagatacac cggctggggc cagctgagcc 2941 ggaagctgat caacggcatc cgggacaagc agtccggcaa gacaatcctg gatttcctga 3001 agtccgacgg cttcgccaac agaaacttca tgcagctgat ccacgacgac agcctgacct 3061 ttaaagagga catccagaaa gcccaggtgt ccggccaggg cgatagcctg cacgagcaca 3121 ttgccaatct ggccggcagc cccgccatta agaagggcat cctgcagaca gtgaaggtgg 3181 tggacgagct cgtgaaagtg atgggccggc acaagcccga gaacatcgtg atcgaaatgg 3241 ccagagagaa ccagaccacc cagaagggac agaagaacag ccgcgagaga atgaagcgga 3301 tcgaagaggg catcaaagag ctgggcagcc agatcctgaa agaacacccc gtggaaaaca 3361 cccagctgca gaacgagaag ctgtacctgt actacctgca gaatgggcgg gatatgtacg 3421 tggaccagga actggacatc aaccggctgt ccgactacga tgtggaccat atcgtgcctc 3481 agagctttct gaaggacgac tccatcgaca acaaggtgct gaccagaagc gacaagaacc 3541 ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa gaagatgaag aactactggc 3601 ggcagctgct gaacgccaag ctgattaccc agagaaagtt cgacaatctg accaaggccg 3661 agagaggcgg cctgagcgaa ctggataagg ccggcttcat caagagacag ctggtggaaa 3721 cccggcagat cacaaagcac gtggcacaga tcctggactc ccggatgaac actaagtacg 3781 acgagaatga caagctgatc cgggaagtga aagtgatcac cctgaagtcc aagctggtgt 3841 ccgatttccg gaaggatttc cagttttaca aagtgcgcga gatcaacaac taccaccacg 3901 cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag taccctaagc 3961 tggaaagcga gttcgtgtac ggcgactaca aggtgtacga cgtgcggaag atgatcgcca 4021 agagcgagca ggaaatcggc aaggctaccg ccaagtactt cttctacagc aacatcatga 4081 actttttcaa gaccgagatt accctggcca acggcgagat ccggaagcgg cctctgatcg 4141 agacaaacgg cgaaaccggg gagatcgtgt gggataaggg ccgggatttt gccaccgtgc 4201 ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa gaccgaggtg cagacaggcg 4261 gcttcagcaa agagtctatc ctgcccaaga ggaacagcga taagctgatc gccagaaaga 4321 aggactggga ccctaagaag tacggcggct tcgacagccc caccgtggcc tattctgtgc 4381 tggtggtggc caaagtggaa aagggcaagt ccaagaaact gaagagtgtg aaagagctgc 4441 tggggatcac catcatggaa agaagcagct tcgagaagaa tcccatcgac tttctggaag 4501 ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa gctgcctaag tactccctgt 4561 tcgagctgga aaacggccgg aagagaatgc tggcctctgc cggcgaactg cagaagggaa 4621 acgaactggc cctgccctcc aaatatgtga acttcctgta cctggccagc cactatgaga 4681 agctgaaggg ctcccccgag gataatgagc agaaacagct gtttgtggaa cagcacaagc 4741 actacctgga cgagatcatc gagcagatca gcgagttctc caagagagtg atcctggccg 4801 acgctaatct ggacaaagtg ctgtccgcct acaacaagca ccgggataag cccatcagag 4861 agcaggccga gaatatcatc cacctgttta ccctgaccaa tctgggagcc cctgccgcct 4921 tcaagtactt tgacaccacc atcgaccgga agaggtacac cagcaccaaa gaggtgctgg 4981 acgccaccct gatccaccag agcatcaccg gcctgtacga gacacggatc gacctgtctc 5041 agctgggagg cgacaaaagg ccggcggcca cgaaaaaggc cggccaggca aaaaagaaaa 5101 agtaagaatt cctagagctc gctgatcagc ctcgactgtg ccttctagtt gccagccatc 5161 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct 5221 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg 5281 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gagaatagca ggcatgctgg 5341 ggagcggccg caggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc 5401 gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc 5461 agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt ttctccttac 5521 gcatctgtgc ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc 5581 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc 5641 gccttagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt 5701 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac 5761 ctcgacccca aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag 5821 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa 5881 actggaacaa cactcaactc tatctcgggc tattcttttg atttataagg gattttgccg 5941 atttcggtct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac 6001 aaaatattaa cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca 6061 tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg 6121 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg 6181 ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta 6241 taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat 6301 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg 6361 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa 6421 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac 6481 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac 6541 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt 6601 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc 6661 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca 6721 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc 6781 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag 6841 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa 6901 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg 6961 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa 7021 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg 7081 gctggctggt ttattgctga taaatctgga gccggtgagc gtggaagccg cggtatcatt 7141 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt 7201 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag 7261 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat 7321 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct 7381 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct 7441 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca 7501 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc 7561 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc 7621 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct 7681 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag 7741 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc 7801 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg 7861 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag 7921 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt 7981 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac 8041 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgt //