LOCUS Exported 7615 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Expresses dSp Cas9 fused to truncated p65 activation domain. ACCESSION . VERSION . KEYWORDS dSp-p65 (1-100) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7615) TITLE Church Lab CRISPR plasmids 2017 REFERENCE 2 (bases 1 to 7615) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7615 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 146..525 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 526..729 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 680..700 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 726..750 /label=LNCX /note="Human CMV promoter, forward primer" regulatory 838..847 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 844..4947 /codon_start=1 /product="catalytically dead mutant of the Cas9 endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /label=Cas9m4 /note="RNA-guided DNA-binding protein that lacks endonuclease activity due to the mutations D10A, D839A, H840A, and N863A (Mali et al., 2013)" /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY LYYLQNGRDMYVDQELDINRLSDYDVAAIVPQSFLKDDSIDNKVLTRSDKARGKSDNVP SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL DATLIHQSITGLYETRIDLSQLGGD" CDS 4960..4980 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" primer_bind complement(5365..5384) /label=TK-pA-R /note="Thymidine kinase polyA, reverse primer" polyA_signal 5409..5457 /label=HSV TK poly(A) signal /note="herpes simplex virus thymidine kinase polyadenylation signal (Cole and Stacy, 1985)" rep_origin complement(5771..6359) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5851..5870) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6530..7390) /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 7153..7172 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7391..7495) /gene="bla" /label=AmpR promoter primer_bind complement(7570..7589) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" ORIGIN 1 gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa 61 ggcttgaccg acaattgcat gaagaatctg cttagggtta ggcgttttgc gctgcttcgc 121 gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta atagtaatca 181 attacggggt cattagttca tagcccatat atggagttcc gcgttacata acttacggta 241 aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat 301 gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg 361 taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac 421 gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt 481 cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg 541 cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc 601 attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt 661 aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata 721 agcagagctc gtttagtgaa ccgtcagatc gcctggagac gccatccacg ctgttttgac 781 ctccatagaa gacaccggga ccgatccagc ctccggactc tagaggatcg aacccttgcc 841 accatggaca agaagtactc cattgggctc gctatcggca caaacagcgt cggctgggcc 901 gtcattacgg acgagtacaa ggtgccgagc aaaaaattca aagttctggg caataccgat 961 cgccacagca taaagaagaa cctcattggc gccctcctgt tcgactccgg ggagacggcc 1021 gaagccacgc ggctcaaaag aacagcacgg cgcagatata cccgcagaaa gaatcggatc 1081 tgctacctgc aggagatctt tagtaatgag atggctaagg tggatgactc tttcttccat 1141 aggctggagg agtccttttt ggtggaggag gataaaaagc acgagcgcca cccaatcttt 1201 ggcaatatcg tggacgaggt ggcgtaccat gaaaagtacc caaccatata tcatctgagg 1261 aagaagcttg tagacagtac tgataaggct gacttgcggt tgatctatct cgcgctggcg 1321 catatgatca aatttcgggg acacttcctc atcgaggggg acctgaaccc agacaacagc 1381 gatgtcgaca aactctttat ccaactggtt cagacttaca atcagctttt cgaagagaac 1441 ccgatcaacg catccggagt tgacgccaaa gcaatcctga gcgctaggct gtccaaatcc 1501 cggcggctcg aaaacctcat cgcacagctc cctggggaga agaagaacgg cctgtttggt 1561 aatcttatcg ccctgtcact cgggctgacc cccaacttta aatctaactt cgacctggcc 1621 gaagatgcca agcttcaact gagcaaagac acctacgatg atgatctcga caatctgctg 1681 gcccagatcg gcgaccagta cgcagacctt tttttggcgg caaagaacct gtcagacgcc 1741 attctgctga gtgatattct gcgagtgaac acggagatca ccaaagctcc gctgagcgct 1801 agtatgatca agcgctatga tgagcaccac caagacttga ctttgctgaa ggcccttgtc 1861 agacagcaac tgcctgagaa gtacaaggaa attttcttcg atcagtctaa aaatggctac 1921 gccggataca ttgacggcgg agcaagccag gaggaatttt acaaatttat taagcccatc 1981 ttggaaaaaa tggacggcac cgaggagctg ctggtaaagc ttaacagaga agatctgttg 2041 cgcaaacagc gcactttcga caatggaagc atcccccacc agattcacct gggcgaactg 2101 cacgctatcc tcaggcggca agaggatttc tacccctttt tgaaagataa cagggaaaag 2161 attgagaaaa tcctcacatt tcggataccc tactatgtag gccccctcgc ccggggaaat 2221 tccagattcg cgtggatgac tcgcaaatca gaagagacca tcactccctg gaacttcgag 2281 gaagtcgtgg ataagggggc ctctgcccag tccttcatcg aaaggatgac taactttgat 2341 aaaaatctgc ctaacgaaaa ggtgcttcct aaacactctc tgctgtacga gtacttcaca 2401 gtttataacg agctcaccaa ggtcaaatac gtcacagaag ggatgagaaa gccagcattc 2461 ctgtctggag agcagaagaa agctatcgtg gacctcctct tcaagacgaa ccggaaagtt 2521 accgtgaaac agctcaaaga agactatttc aaaaagattg aatgtttcga ctctgttgaa 2581 atcagcggag tggaggatcg cttcaacgca tccctgggaa cgtatcacga tctcctgaaa 2641 atcattaaag acaaggactt cctggacaat gaggagaacg aggacattct tgaggacatt 2701 gtcctcaccc ttacgttgtt tgaagatagg gagatgattg aagaacgctt gaaaacttac 2761 gctcatctct tcgacgacaa agtcatgaaa cagctcaaga ggcgccgata tacaggatgg 2821 gggcggctgt caagaaaact gatcaatggg atccgagaca agcagagtgg aaagacaatc 2881 ctggattttc ttaagtccga tggatttgcc aaccggaact tcatgcagtt gatccatgat 2941 gactctctca cctttaagga ggacatccag aaagcacaag tttctggcca gggggacagt 3001 cttcacgagc acatcgctaa tcttgcaggt agcccagcta tcaaaaaggg aatactgcag 3061 accgttaagg tcgtggatga actcgtcaaa gtaatgggaa ggcataagcc cgagaatatc 3121 gttatcgaga tggcccgaga gaaccaaact acccagaagg gacagaagaa cagtagggaa 3181 aggatgaaga ggattgaaga gggtataaaa gaactggggt cccaaatcct taaggaacac 3241 ccagttgaaa acacccagct tcagaatgag aagctctacc tgtactacct gcagaacggc 3301 agggacatgt acgtggatca ggaactggac atcaatcggc tctccgacta cgacgtggct 3361 gctatcgtgc cccagtcttt tctcaaagat gattctattg ataataaagt gttgacaaga 3421 tccgataaag ctagagggaa gagtgataac gtcccctcag aagaagttgt caagaaaatg 3481 aaaaattatt ggcggcagct gctgaacgcc aaactgatca cacaacggaa gttcgataat 3541 ctgactaagg ctgaacgagg tggcctgtct gagttggata aagccggctt catcaaaagg 3601 cagcttgttg agacacgcca gatcaccaag cacgtggccc aaattctcga ttcacgcatg 3661 aacaccaagt acgatgaaaa tgacaaactg attcgagagg tgaaagttat tactctgaag 3721 tctaagctgg tctcagattt cagaaaggac tttcagtttt ataaggtgag agagatcaac 3781 aattaccacc atgcgcatga tgcctacctg aatgcagtgg taggcactgc acttatcaaa 3841 aaatatccca agcttgaatc tgaatttgtt tacggagact ataaagtgta cgatgttagg 3901 aaaatgatcg caaagtctga gcaggaaata ggcaaggcca ccgctaagta cttcttttac 3961 agcaatatta tgaatttttt caagaccgag attacactgg ccaatggaga gattcggaag 4021 cgaccactta tcgaaacaaa cggagaaaca ggagaaatcg tgtgggacaa gggtagggat 4081 ttcgcgacag tccggaaggt cctgtccatg ccgcaggtga acatcgttaa aaagaccgaa 4141 gtacagaccg gaggcttctc caaggaaagt atcctcccga aaaggaacag cgacaagctg 4201 atcgcacgca aaaaagattg ggaccccaag aaatacggcg gattcgattc tcctacagtc 4261 gcttacagtg tactggttgt ggccaaagtg gagaaaggga agtctaaaaa actcaaaagc 4321 gtcaaggaac tgctgggcat cacaatcatg gagcgatcaa gcttcgaaaa aaaccccatc 4381 gactttctcg aggcgaaagg atataaagag gtcaaaaaag acctcatcat taagcttccc 4441 aagtactctc tctttgagct tgaaaacggc cggaaacgaa tgctcgctag tgcgggcgag 4501 ctgcagaaag gtaacgagct ggcactgccc tctaaatacg ttaatttctt gtatctggcc 4561 agccactatg aaaagctcaa agggtctccc gaagataatg agcagaagca gctgttcgtg 4621 gaacaacaca aacactacct tgatgagatc atcgagcaaa taagcgaatt ctccaaaaga 4681 gtgatcctcg ccgacgctaa cctcgataag gtgctttctg cttacaataa gcacagggat 4741 aagcccatca gggagcaggc agaaaacatt atccacttgt ttactctgac caacttgggc 4801 gcgcctgcag ccttcaagta cttcgacacc accatagaca gaaagcggta cacctctaca 4861 aaggaggtcc tggacgccac actgattcat cagtcaatta cggggctcta tgaaacaaga 4921 atcgacctct ctcagctcgg tggagacagc agggctgacc ccaagaagaa gaggaaggtg 4981 agtggtggag gaagtggcgg gtcagggtcg agccagtacc tgcccgacac cgacgaccgg 5041 caccggatcg aggaaaagcg gaagcggacc tacgagacat tcaagagcat catgaagaag 5101 tcccccttca gcggccccac cgaccctaga cctccaccta gaagaatcgc cgtgcccagc 5161 agatccagcg ccagcgtgcc aaaacctgcc ccccagcctt accccttcac cagcagcctg 5221 agcaccatca actacgacga gttccctacc atggtgttcc ccagcggcca gatctctcag 5281 gcctctgctc tggctccagc ccctcctcag tagaagaatt cgatccctac cggttagtaa 5341 tgagtttaaa cgggggaggc taactgaaac acggaaggag acaataccgg aaggaacccg 5401 cgctatgacg gcaataaaaa gacagaataa aacgcacggg tgttgggtcg tttgttcata 5461 aacgcggggt tcggtcccag ggctggcact ctgtcgatac cccaccgaga ccccattggg 5521 gccaatacgc ccgcgtttct tccttttccc caccccaccc cccaagttcg ggtgaaggcc 5581 cagggctcgc agccaacgtc ggggcggcag gccctccata gtcggtcgtt cggctgcggc 5641 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg 5701 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt 5761 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa 5821 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct 5881 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc 5941 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg 6001 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct 6061 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag 6121 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga 6181 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga 6241 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg 6301 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag 6361 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag 6421 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat 6481 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct 6541 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac 6601 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa 6661 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg 6721 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt 6781 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca 6841 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt 6901 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct 6961 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg 7021 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg 7081 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg 7141 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa 7201 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt 7261 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt 7321 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt 7381 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca 7441 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat 7501 ttccccgaaa agtgccacct gacgtcgacg gatcgggaga tctcccgatc ccctatggtg 7561 cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatctgc tccct //