LOCUS Exported 8768 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expresses wild type SpCas9 fused to ZFP-TS2 in mammalian cells. ACCESSION . VERSION . KEYWORDS pCSDest2-SpCas9-WT-NLS-3XHA-NLS-ZFP_TS2 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8768) AUTHORS Bolukbasi MF, Gupta A, Oikemus S, Derr AG, Garber M, Brodsky MH, Zhu LJ, Wolfe SA TITLE DNA-binding-domain fusions enhance the targeting range and precision of Cas9. JOURNAL Nat Methods. 2015 Oct 19. doi: 10.1038/nmeth.3624. PUBMED 26480473 REFERENCE 2 (bases 1 to 8768) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8768 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 37..55 /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind 37..54 /label=SP6 /note="SP6 promoter, forward primer" protein_bind 110..134 /gene="mutant version of attB" /label=attB1 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" regulatory 138..147 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 144..4247 /codon_start=1 /product="Cas9 (Csn1) endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /label=Cas9 /note="generates RNA-guided double strand breaks in DNA" /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL DATLIHQSITGLYETRIDLSQLGGD" CDS 4260..4280 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 4281..4370 /codon_start=1 /product="three tandem HA epitope tags" /label=3xHA /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA" CDS 4383..4409 /codon_start=1 /product="nuclear localization signal (Makkerh et al., 1996)" /label=NLS /translation="PAAKKKKLD" promoter complement(4797..4814) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" polyA_signal 4819..4953 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(4843..4862) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 4897..4916 /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind complement(5066..5086) /label=T3 /note="T3 promoter, forward primer" promoter complement(5066..5084) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(5105..5121) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5105..5121) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(5118..5140) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 5129..5145 /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(5153..5183) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5198..5219 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(5336..5353) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5507..6095) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5587..5606) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6266..7126) /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 6889..6908 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7127..7231) /gene="bla" /label=AmpR promoter rep_origin complement(7257..7712) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(7394..7415) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 7606..7625 /label=F1ori-R /note="F1 origin, reverse primer" promoter 7786..2 /label=CMV IE94 promoter /note="enhancer/promoter region of simian cytomegalovirus major immediate early transcription unit IE94" ORIGIN 1 accgccattc tgcctgggga cgtcggagca agcttgattt aggtgacact atagaataca 61 agctacttgt tctttttgca ggatcccatc gattcgaatt caaggatcaa caagtttgta 121 caaaaaagca ggctggcgcc accatggaca agaagtactc cattgggctc gatatcggta 181 ccaacagcgt cggctgggcc gtcattacgg acgagtacaa ggtgccgagc aaaaaattca 241 aagttctggg caataccgat cgccacagca taaagaagaa cctcattgga gccctcctgt 301 tcgactccgg ggagacggcc gaagccacgc ggctcaaaag aacagcacgg cgcagatata 361 cccgcagaaa gaatcggatc tgctacctgc aggagatctt tagtaatgag atggctaagg 421 tggatgactc tttcttccat aggctggagg agtccttttt ggtggaggag gataaaaagc 481 acgagcgcca cccaatcttt ggcaatatcg tggacgaggt ggcgtaccat gaaaagtacc 541 caaccatata tcatctgagg aagaagctgg tagacagtac tgataaggct gacttgcggt 601 tgatctatct cgcgctggcg cacatgatca aatttcgggg acacttcctc atcgaggggg 661 acctgaaccc agacaacagc gatgtcgaca aactctttat ccaactggtt cagacttaca 721 atcagctttt cgaggagaac ccgatcaacg catccggcgt tgacgccaaa gcaatcctga 781 gcgctaggct gtccaaatcc cggcggctcg aaaacctcat cgcacagctc cctggggaga 841 agaagaacgg cctgtttggt aatcttatcg ccctgtcact cgggctgacc cccaacttta 901 aatctaactt cgacctggcc gaagatgcca agctgcaact gagcaaagac acctacgatg 961 atgatctcga caatctgctg gcccagatcg gcgaccagta cgcagacctt tttttggcgg 1021 caaagaacct gtcagacgcc attctgctga gtgatattct gcgagtgaac acggagatca 1081 ccaaagctcc gctgagcgct agtatgatca agcgctatga tgagcaccac caagacttga 1141 ctttgctgaa ggcccttgtc agacagcaac tgcctgagaa gtacaaggaa attttcttcg 1201 atcagtctaa aaatggctac gccggataca ttgacggcgg agcaagccag gaggaatttt 1261 acaaatttat taagcccatc ttggaaaaaa tggacggcac cgaggagctg ctggtaaagc 1321 tgaacagaga agatctgttg cgcaaacagc gcactttcga caatggaagc atcccccacc 1381 agattcacct gggcgaactg cacgctatcc tcaggcggca agaggatttc tacccctttt 1441 tgaaagataa cagggaaaag attgagaaaa tcctcacatt tcggataccc tactatgtag 1501 gccccctcgc tcggggaaat tccagattcg cgtggatgac tcgcaaatca gaagagacca 1561 tcactccctg gaacttcgag gaagtcgtgg ataagggggc ctctgcccag tccttcatcg 1621 aaaggatgac taactttgat aaaaatctgc ctaacgaaaa ggtgcttcct aaacactctc 1681 tgctgtacga gtacttcaca gtttataacg agctcaccaa ggtcaaatac gtcacagaag 1741 ggatgagaaa gccagcattc ctgtctggag agcagaagaa agctatcgtg gacctcctct 1801 tcaagacgaa ccggaaagtt accgtgaaac agctcaaaga agactatttc aaaaagattg 1861 aatgtttcga ctctgttgaa atcagcggag tggaggatcg cttcaacgca tccctgggaa 1921 cgtatcacga tctcctgaaa atcattaaag acaaggactt cctggacaat gaggagaacg 1981 aggacattct tgaggacatt gtcctcaccc ttacgttgtt tgaagatagg gagatgattg 2041 aagaacgctt gaaaacttac gctcatctct tcgacgacaa agtcatgaaa cagctcaaga 2101 gacgccgata tacaggatgg gggcggctgt caagaaaact gatcaatggc atccgagaca 2161 agcagagtgg aaagacaatc ctggattttc ttaagtccga tggatttgcc aaccggaact 2221 tcatgcagtt gatccatgat gactctctca cctttaagga ggacatccag aaagcacaag 2281 tttctggcca gggggacagt cttcacgagc acatcgctaa tcttgcaggt agcccagcta 2341 tcaaaaaggg aatactgcag accgttaagg tcgtggatga actcgtcaaa gtaatgggaa 2401 ggcataagcc cgagaatatc gttatcgaga tggcccgaga gaaccaaact acccagaagg 2461 gacagaagaa cagtagggaa aggatgaaga ggattgaaga gggtataaaa gaactggggt 2521 cccaaatcct taaggaacac ccagttgaaa acacccagct tcagaatgag aagctctacc 2581 tgtactacct gcagaacggc agggacatgt acgtggatca ggaactggac atcaaccggt 2641 tgtccgacta cgacgtggat catatcgtgc cccaaagctt tctcaaagat gattctattg 2701 ataataaagt gttgacaaga tccgataaaa atagagggaa gagtgataac gtcccctcag 2761 aagaagttgt caagaaaatg aaaaattatt ggcggcagct gctgaacgcc aaactgatca 2821 cacaacggaa gttcgataat ctgactaagg ctgaacgagg tggcctgtct gagttggata 2881 aagccggctt catcaaaagg cagcttgttg agacacgcca gatcaccaag cacgtggccc 2941 aaattctcga ttcacgcatg aacaccaagt acgatgaaaa tgacaaactg attcgagagg 3001 tgaaagttat tactctgaag tctaagctgg tctcagattt cagaaaggac tttcagtttt 3061 ataaggtgag agagatcaac aattaccacc atgcgcatga tgcctacctg aatgcagtgg 3121 taggcactgc acttatcaaa aaatatccca agctggaatc tgaatttgtt tacggagact 3181 ataaagtgta cgatgttagg aaaatgatcg caaagtctga gcaggaaata ggcaaggcca 3241 ccgctaagta cttcttttac agcaatatta tgaatttttt caagaccgag attacactgg 3301 ccaatggaga gattcggaag cgaccactta tcgaaacaaa cggagaaaca ggagaaatcg 3361 tgtgggacaa gggtagggat ttcgcgacag tccgcaaggt cctgtccatg ccgcaggtga 3421 acatcgttaa aaagaccgaa gtacagaccg gaggcttctc caaggaaagt atcctcccga 3481 aaaggaacag cgacaagctg atcgcacgca aaaaagattg ggaccccaag aaatacggcg 3541 gattcgattc tcctacagtc gcttacagtg tactggttgt ggccaaagtg gagaaaggga 3601 agtctaaaaa actcaaaagc gtcaaggaac tgctgggcat cacaatcatg gagcgatcca 3661 gcttcgagaa aaaccccatc gactttctcg aagcgaaagg atataaagag gtcaaaaaag 3721 acctcatcat taagctgccc aagtactctc tctttgagct tgaaaacggc cggaaacgaa 3781 tgctcgctag tgcgggcgag ctgcagaaag gtaacgagct ggcactgccc tctaaatacg 3841 ttaatttctt gtatctggcc agccactatg aaaagctcaa agggtctccc gaagataatg 3901 agcagaagca gctgttcgtg gaacaacaca aacactacct tgatgagatc atcgagcaaa 3961 taagcgagtt ctccaaaaga gtgatcctcg ccgacgctaa cctcgataag gtgctttctg 4021 cttacaataa gcacagggat aagcccatca gggagcaggc agaaaacatt atccacttgt 4081 ttactctgac caacttgggc gcgcctgcag ccttcaagta cttcgacacc accatagaca 4141 gaaagcggta cacctctaca aaggaggtcc tggacgccac actgattcat cagtcaatta 4201 cggggctcta tgaaacaaga atcgacctct ctcagctcgg tggagacggc accggcgggc 4261 ccaagaagaa gaggaaggta tacccatacg atgttcctga ctatgcgggc tatccctatg 4321 acgtcccgga ctatgcagga tcgtatcctt atgacgttcc agattacgct ggatccgccg 4381 ctccggcagc taagaaaaag aaactggatt tcgaatccgg aaagccctat aaatgtcctg 4441 aatgtggcaa gtccttctcg gagaagagcc acctgacacg gcaccaacgc acgcacactg 4501 gtgagaagcc atacgcgtgt cctgtcgagt cctgtgaccg ccgcttcagt cggagcgacc 4561 acctgacaca gcacatccgc attcacacag ggcaaaaacc gtttcaatgc cgcatctgca 4621 tgaggaactt cagcgacaag ggccacctga cccggcacat ccgcacccac acaggagaaa 4681 agcccttcgc ctgtgacatc tgcggcagga agttcgcgcg gagcgacgac ctgacacggc 4741 acaccaagat ccacctccgt cagaaagacc ccgggtaatg actcgagcct ctagaactat 4801 agtgagtcgt attacgtaga tccagacatg ataagataca ttgatgagtt tggacaaacc 4861 acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta 4921 tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg 4981 tttcaggttc agggggaggt gtgggaggtt ttttaattcg cggccgcggc gccaatgcat 5041 tgggcccggt acccagcttt tgttcccttt agtgagggtt aattgcgcgc ttggcgtaat 5101 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac 5161 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa 5221 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat 5281 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc 5341 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg 5401 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag 5461 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc 5521 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag 5581 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga 5641 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc 5701 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg 5761 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt 5821 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca 5881 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca 5941 ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag 6001 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca 6061 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg 6121 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa 6181 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta 6241 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag 6301 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga 6361 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac 6421 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc 6481 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta 6541 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac 6601 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat 6661 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa 6721 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg 6781 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag 6841 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc 6901 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct 6961 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat 7021 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg 7081 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc 7141 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta 7201 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctaaat 7261 tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt 7321 taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg 7381 gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt 7441 caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc 7501 aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg 7561 atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa 7621 aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc 7681 cgccgcgctt aatgcgccgc tacagggcgc gtcccattcg ccattcaggc tgcgcaactg 7741 ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagtcgacca tagccaattc 7801 aatatggcgt atatggactc atgccaattc aatatggtgg atctggacct gtgccaattc 7861 aatatggcgt atatggactc gtgccaattc aatatggtgg atctggaccc cagccaattc 7921 aatatggcgg acttggcacc atgccaattc aatatggcgg acttggcact gtgccaactg 7981 gggaggggtc tacttggcac ggtgccaagt ttgaggaggg gtcttggccc tgtgccaagt 8041 ccgccatatt gaattggcat ggtgccaata atggcggcca tattggctat atgccaggat 8101 caatatatag gcaatatcca atatggccct atgccaatat ggctattggc caggttcaat 8161 actatgtatt ggccctatgc catatagtat tccatatatg ggttttccta ttgacgtaga 8221 tagcccctcc caatgggcgg tcccatatac catatatggg gcttcctaat accgcccata 8281 gccactcccc cattgacgtc aatggtctct atatatggtc tttcctattg acgtcatatg 8341 ggcggtccta ttgacgtata tggcgcctcc cccattgacg tcaattacgg taaatggccc 8401 gcctggctca atgcccattg acgtcaatag gaccacccac cattgacgtc aatgggatgg 8461 ctcattgccc attcatatcc gttctcacgc cccctattga cgtcaatgac ggtaaatggc 8521 ccacttggca gtacatcaat atctattaat agtaacttgg caagtacatt actattggaa 8581 ggacgccagg gtacattggc agtactccca ttgacgtcaa tggcggtaaa tggcccgcga 8641 tggctgccaa gtacatcccc attgacgtca atggggaggg gcaatgacgc aaatgggcgt 8701 tccattgacg taaatgggcg gtaggcgtgc ctaatgggag gtctatataa gcaatgctcg 8761 tttaggga //