LOCUS Exported 7614 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Human expression plasmid for SpCas9 Cluster 1 variant (HypaCas9): CMV-T7-hSpCas9-Cluster1(N692A, M694A, Q695A, H698A)-NLS(SV40)-3xFLAG. ACCESSION . VERSION . KEYWORDS BPK4410 - human expression plasmid for SpCas9 Cluster 1 (HypaC... SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7614) AUTHORS Chen JS, Dagdas YS, Kleinstiver BP, Welch MM, Sousa AA, Harrington LB, Sternberg SH, Joung JK, Yildiz A, Doudna JA TITLE Enhanced proofreading governs CRISPR-Cas9 targeting accuracy. JOURNAL Nature. 2017 Sep 20. doi: 10.1038/nature24268. PUBMED 28931002 REFERENCE 2 (bases 1 to 7614) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7614 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(46..65) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 237..616 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 617..820 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 771..791 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 862..881 /label=T7 /note="T7 promoter, forward primer" promoter 862..880 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" regulatory 888..897 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 894..4997 /codon_start=1 /product="Cas9 endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system, mutated to improve targeting specificity without compromising on-target activity (Chen et al., 2017)" /label=HypaCas9 /note="carries the mutations N692A, M694A, Q695A, and H698A" /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRAFA ALIADDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL DATLIHQSITGLYETRIDLSQLGGD" CDS 5004..5024 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 5031..5096 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 5114..5131 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(5154..5171) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 5160..5384 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(5455..5471) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5455..5471) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(5468..5490) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 5479..5495 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." promoter complement(5503..5533) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5548..5569 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(5686..5703) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5857..6445) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5937..5956) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6616..7476) /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 7239..7258 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7477..7581) /gene="bla" /label=AmpR promoter ORIGIN 1 cggatcggga gatcgatctc ccgatcccct agggtcgact ctcagtacaa tctgctctga 61 tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg 121 cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct 181 gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgttgaca 241 ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc atagcccata 301 tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga 361 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt 421 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt 481 gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca 541 ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt 601 catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt 661 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca 721 ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg 781 cggtaggcgt gtacggtggg aggtctatat aagcagagct ggtttagtga accgtcagat 841 ccgctagaga tccgcggccg ctaatacgac tcactatagg gagagccgcc accatggata 901 aaaagtattc tattggttta gacatcggca ctaattccgt tggatgggct gtcataaccg 961 atgaatacaa agtaccttca aagaaattta aggtgttggg gaacacagac cgtcattcga 1021 ttaaaaagaa tcttatcggt gccctcctat tcgatagtgg cgaaacggca gaggcgactc 1081 gcctgaaacg aaccgctcgg agaaggtata cacgtcgcaa gaaccgaata tgttacttac 1141 aagaaatttt tagcaatgag atggccaaag ttgacgattc tttctttcac cgtttggaag 1201 agtccttcct tgtcgaagag gacaagaaac atgaacggca ccccatcttt ggaaacatag 1261 tagatgaggt ggcatatcat gaaaagtacc caacgattta tcacctcaga aaaaagctag 1321 ttgactcaac tgataaagcg gacctgaggt taatctactt ggctcttgcc catatgataa 1381 agttccgtgg gcactttctc attgagggtg atctaaatcc ggacaactcg gatgtcgaca 1441 aactgttcat ccagttagta caaacctata atcagttgtt tgaagagaac cctataaatg 1501 caagtggcgt ggatgcgaag gctattctta gcgcccgcct ctctaaatcc cgacggctag 1561 aaaacctgat cgcacaatta cccggagaga agaaaaatgg gttgttcggt aaccttatag 1621 cgctctcact aggcctgaca ccaaatttta agtcgaactt cgacttagct gaagatgcca 1681 aattgcagct tagtaaggac acgtacgatg acgatctcga caatctactg gcacaaattg 1741 gagatcagta tgcggactta tttttggctg ccaaaaacct tagcgatgca atcctcctat 1801 ctgacatact gagagttaat actgagatta ccaaggcgcc gttatccgct tcaatgatca 1861 aaaggtacga tgaacatcac caagacttga cacttctcaa ggccctagtc cgtcagcaac 1921 tgcctgagaa atataaggaa atattctttg atcagtcgaa aaacgggtac gcaggttata 1981 ttgacggcgg agcgagtcaa gaggaattct acaagtttat caaacccata ttagagaaga 2041 tggatgggac ggaagagttg cttgtaaaac tcaatcgcga agatctactg cgaaagcagc 2101 ggactttcga caacggtagc attccacatc aaatccactt aggcgaattg catgctatac 2161 ttagaaggca ggaggatttt tatccgttcc tcaaagacaa tcgtgaaaag attgagaaaa 2221 tcctaacctt tcgcatacct tactatgtgg gacccctggc ccgagggaac tctcggttcg 2281 catggatgac aagaaagtcc gaagaaacga ttactccatg gaattttgag gaagttgtcg 2341 ataaaggtgc gtcagctcaa tcgttcatcg agaggatgac caactttgac aagaatttac 2401 cgaacgaaaa agtattgcct aagcacagtt tactttacga gtatttcaca gtgtacaatg 2461 aactcacgaa agttaagtat gtcactgagg gcatgcgtaa acccgccttt ctaagcggag 2521 aacagaagaa agcaatagta gatctgttat tcaagaccaa ccgcaaagtg acagttaagc 2581 aattgaaaga ggactacttt aagaaaattg aatgcttcga ttctgtcgag atctccgggg 2641 tagaagatcg atttaatgcg tcacttggta cgtatcatga cctcctaaag ataattaaag 2701 ataaggactt cctggataac gaagagaatg aagatatctt agaagatata gtgttgactc 2761 ttaccctctt tgaagatcgg gaaatgattg aggaaagact aaaaacatac gctcacctgt 2821 tcgacgataa ggttatgaaa cagttaaaga ggcgtcgcta tacgggctgg ggacgattgt 2881 cgcggaaact tatcaacggg ataagagaca agcaaagtgg taaaactatt ctcgattttc 2941 taaagagcga cggcttcgcc aatagggcct ttgcagccct gatcgctgat gactctttaa 3001 ccttcaaaga ggatatacaa aaggcacagg tttccggaca aggggactca ttgcacgaac 3061 atattgcgaa tcttgctggt tcgccagcca tcaaaaaggg catactccag acagtcaaag 3121 tagtggatga gctagttaag gtcatgggac gtcacaaacc ggaaaacatt gtaatcgaga 3181 tggcacgcga aaatcaaacg actcagaagg ggcaaaaaaa cagtcgagag cggatgaaga 3241 gaatagaaga gggtattaaa gaactgggca gccagatctt aaaggagcat cctgtggaaa 3301 atacccaatt gcagaacgag aaactttacc tctattacct acaaaatgga agggacatgt 3361 atgttgatca ggaactggac ataaaccgtt tatctgatta cgacgtcgat cacattgtac 3421 cccaatcctt tttgaaggac gattcaatcg acaataaagt gcttacacgc tcggataaga 3481 accgagggaa aagtgacaat gttccaagcg aggaagtcgt aaagaaaatg aagaactatt 3541 ggcggcagct cctaaatgcg aaactgataa cgcaaagaaa gttcgataac ttaactaaag 3601 ctgagagggg tggcttgtct gaacttgaca aggccggatt tattaaacgt cagctcgtgg 3661 aaacccgcca aatcacaaag catgttgcac agatactaga ttcccgaatg aatacgaaat 3721 acgacgagaa cgataagctg attcgggaag tcaaagtaat cactttaaag tcaaaattgg 3781 tgtcggactt cagaaaggat tttcaattct ataaagttag ggagataaat aactaccacc 3841 atgcgcacga cgcttatctt aatgccgtcg tagggaccgc actcattaag aaatacccga 3901 agctagaaag tgagtttgtg tatggtgatt acaaagttta tgacgtccgt aagatgatcg 3961 cgaaaagcga acaggagata ggcaaggcta cagccaaata cttcttttat tctaacatta 4021 tgaatttctt taagacggaa atcactctgg caaacggaga gatacgcaaa cgacctttaa 4081 ttgaaaccaa tggggagaca ggtgaaatcg tatgggataa gggccgggac ttcgcgacgg 4141 tgagaaaagt tttgtccatg ccccaagtca acatagtaaa gaaaactgag gtgcagaccg 4201 gagggttttc aaaggaatcg attcttccaa aaaggaatag tgataagctc atcgctcgta 4261 aaaaggactg ggacccgaaa aagtacggtg gcttcgatag ccctacagtt gcctattctg 4321 tcctagtagt ggcaaaagtt gagaagggaa aatccaagaa actgaagtca gtcaaagaat 4381 tattggggat aacgattatg gagcgctcgt cttttgaaaa gaaccccatc gacttccttg 4441 aggcgaaagg ttacaaggaa gtaaaaaagg atctcataat taaactacca aagtatagtc 4501 tgtttgagtt agaaaatggc cgaaaacgga tgttggctag cgccggagag cttcaaaagg 4561 ggaacgaact cgcactaccg tctaaatacg tgaatttcct gtatttagcg tcccattacg 4621 agaagttgaa aggttcacct gaagataacg aacagaagca actttttgtt gagcagcaca 4681 aacattatct cgacgaaatc atagagcaaa tttcggaatt cagtaagaga gtcatcctag 4741 ctgatgccaa tctggacaaa gtattaagcg catacaacaa gcacagggat aaacccatac 4801 gtgagcaggc ggaaaatatt atccatttgt ttactcttac caacctcggc gctccagccg 4861 cattcaagta ttttgacaca acgatagatc gcaaacgata cacttctacc aaggaggtgc 4921 tagacgcgac actgattcac caatccatca cgggattata tgaaactcgg atagatttgt 4981 cacagcttgg gggtgacgga tcccccaaga agaagaggaa agtctcgagc gactacaaag 5041 accatgacgg tgattataaa gatcatgaca tcgattacaa ggatgacgat gacaaggctg 5101 caggatgacc ggtcatcatc accatcacca ttgagtttaa acccgctgat cagcctcgac 5161 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct 5221 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct 5281 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg 5341 ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag 5401 aaccagctgg ggctcgatac cgtcgacctc tagctagagc ttggcgtaat catggtcata 5461 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag 5521 cataaagtgt aaagcctagg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg 5581 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca 5641 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc 5701 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg 5761 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa 5821 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga 5881 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag 5941 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct 6001 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg 6061 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc 6121 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt 6181 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta 6241 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac 6301 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc 6361 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat 6421 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc 6481 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt 6541 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta 6601 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct 6661 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg 6721 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga 6781 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt 6841 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt 6901 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt 6961 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat 7021 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc 7081 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc 7141 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat 7201 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag 7261 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt 7321 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc 7381 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa 7441 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg 7501 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa 7561 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tcga //