LOCUS Exported 8278 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expresses dSp Cas9 fused to combination of truncated activation domains. ACCESSION . VERSION . KEYWORDS dSp-VP64-p65(100-261)-RTA(125-190) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8278) TITLE Church Lab CRISPR plasmids 2017 REFERENCE 2 (bases 1 to 8278) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8278 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 110..489 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 490..693 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 644..664 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 690..714 /label=LNCX /note="Human CMV promoter, forward primer" regulatory 802..811 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 808..4911 /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 4924..4944 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 4999..5148 /codon_start=1 /product="tetrameric repeat of the minimal activation domain of herpes simplex virus VP16 (Beerli et al., 1998)" /label=VP64 /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF DLDML" CDS 5173..5193 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 5353..5709 /codon_start=1 /product="transcriptional activation domain of human RelA, also known as p65 (O'Shea and Perkins, 2008)" /label=RelA (p65) AD /translation="PTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNS EFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGD EDFSSIADMDFSALL" primer_bind complement(5992..6011) /label=TK-pA-R /note="Thymidine kinase polyA, reverse primer" polyA_signal 6036..6084 /label=HSV TK poly(A) signal /note="herpes simplex virus thymidine kinase polyadenylation signal (Cole and Stacy, 1985)" rep_origin complement(6398..6986) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(6478..6497) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(7157..8017) /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 7780..7799 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(8018..8122) /gene="bla" /label=AmpR promoter primer_bind complement(8197..8216) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" ORIGIN 1 aaaatttaag ctacaacaag gcaaggcttg accgacaatt gcatgaagaa tctgcttagg 61 gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg acattgatta 121 ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag 181 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc 241 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga 301 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat 361 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc 421 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct 481 attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca 541 cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat 601 caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg 661 cgtgtacggt gggaggtcta tataagcaga gctcgtttag tgaaccgtca gatcgcctgg 721 agacgccatc cacgctgttt tgacctccat agaagacacc gggaccgatc cagcctccgg 781 actctagagg atcgaaccct tgccaccatg gacaagaagt actccattgg gctcgctatc 841 ggcacaaaca gcgtcggctg ggccgtcatt acggacgagt acaaggtgcc gagcaaaaaa 901 ttcaaagttc tgggcaatac cgatcgccac agcataaaga agaacctcat tggcgccctc 961 ctgttcgact ccggggagac ggccgaagcc acgcggctca aaagaacagc acggcgcaga 1021 tatacccgca gaaagaatcg gatctgctac ctgcaggaga tctttagtaa tgagatggct 1081 aaggtggatg actctttctt ccataggctg gaggagtcct ttttggtgga ggaggataaa 1141 aagcacgagc gccacccaat ctttggcaat atcgtggacg aggtggcgta ccatgaaaag 1201 tacccaacca tatatcatct gaggaagaag cttgtagaca gtactgataa ggctgacttg 1261 cggttgatct atctcgcgct ggcgcatatg atcaaatttc ggggacactt cctcatcgag 1321 ggggacctga acccagacaa cagcgatgtc gacaaactct ttatccaact ggttcagact 1381 tacaatcagc ttttcgaaga gaacccgatc aacgcatccg gagttgacgc caaagcaatc 1441 ctgagcgcta ggctgtccaa atcccggcgg ctcgaaaacc tcatcgcaca gctccctggg 1501 gagaagaaga acggcctgtt tggtaatctt atcgccctgt cactcgggct gacccccaac 1561 tttaaatcta acttcgacct ggccgaagat gccaagcttc aactgagcaa agacacctac 1621 gatgatgatc tcgacaatct gctggcccag atcggcgacc agtacgcaga cctttttttg 1681 gcggcaaaga acctgtcaga cgccattctg ctgagtgata ttctgcgagt gaacacggag 1741 atcaccaaag ctccgctgag cgctagtatg atcaagcgct atgatgagca ccaccaagac 1801 ttgactttgc tgaaggccct tgtcagacag caactgcctg agaagtacaa ggaaattttc 1861 ttcgatcagt ctaaaaatgg ctacgccgga tacattgacg gcggagcaag ccaggaggaa 1921 ttttacaaat ttattaagcc catcttggaa aaaatggacg gcaccgagga gctgctggta 1981 aagcttaaca gagaagatct gttgcgcaaa cagcgcactt tcgacaatgg aagcatcccc 2041 caccagattc acctgggcga actgcacgct atcctcaggc ggcaagagga tttctacccc 2101 tttttgaaag ataacaggga aaagattgag aaaatcctca catttcggat accctactat 2161 gtaggccccc tcgcccgggg aaattccaga ttcgcgtgga tgactcgcaa atcagaagag 2221 accatcactc cctggaactt cgaggaagtc gtggataagg gggcctctgc ccagtccttc 2281 atcgaaagga tgactaactt tgataaaaat ctgcctaacg aaaaggtgct tcctaaacac 2341 tctctgctgt acgagtactt cacagtttat aacgagctca ccaaggtcaa atacgtcaca 2401 gaagggatga gaaagccagc attcctgtct ggagagcaga agaaagctat cgtggacctc 2461 ctcttcaaga cgaaccggaa agttaccgtg aaacagctca aagaagacta tttcaaaaag 2521 attgaatgtt tcgactctgt tgaaatcagc ggagtggagg atcgcttcaa cgcatccctg 2581 ggaacgtatc acgatctcct gaaaatcatt aaagacaagg acttcctgga caatgaggag 2641 aacgaggaca ttcttgagga cattgtcctc acccttacgt tgtttgaaga tagggagatg 2701 attgaagaac gcttgaaaac ttacgctcat ctcttcgacg acaaagtcat gaaacagctc 2761 aagaggcgcc gatatacagg atgggggcgg ctgtcaagaa aactgatcaa tgggatccga 2821 gacaagcaga gtggaaagac aatcctggat tttcttaagt ccgatggatt tgccaaccgg 2881 aacttcatgc agttgatcca tgatgactct ctcaccttta aggaggacat ccagaaagca 2941 caagtttctg gccaggggga cagtcttcac gagcacatcg ctaatcttgc aggtagccca 3001 gctatcaaaa agggaatact gcagaccgtt aaggtcgtgg atgaactcgt caaagtaatg 3061 ggaaggcata agcccgagaa tatcgttatc gagatggccc gagagaacca aactacccag 3121 aagggacaga agaacagtag ggaaaggatg aagaggattg aagagggtat aaaagaactg 3181 gggtcccaaa tccttaagga acacccagtt gaaaacaccc agcttcagaa tgagaagctc 3241 tacctgtact acctgcagaa cggcagggac atgtacgtgg atcaggaact ggacatcaat 3301 cggctctccg actacgacgt ggctgctatc gtgccccagt cttttctcaa agatgattct 3361 attgataata aagtgttgac aagatccgat aaagctagag ggaagagtga taacgtcccc 3421 tcagaagaag ttgtcaagaa aatgaaaaat tattggcggc agctgctgaa cgccaaactg 3481 atcacacaac ggaagttcga taatctgact aaggctgaac gaggtggcct gtctgagttg 3541 gataaagccg gcttcatcaa aaggcagctt gttgagacac gccagatcac caagcacgtg 3601 gcccaaattc tcgattcacg catgaacacc aagtacgatg aaaatgacaa actgattcga 3661 gaggtgaaag ttattactct gaagtctaag ctggtctcag atttcagaaa ggactttcag 3721 ttttataagg tgagagagat caacaattac caccatgcgc atgatgccta cctgaatgca 3781 gtggtaggca ctgcacttat caaaaaatat cccaagcttg aatctgaatt tgtttacgga 3841 gactataaag tgtacgatgt taggaaaatg atcgcaaagt ctgagcagga aataggcaag 3901 gccaccgcta agtacttctt ttacagcaat attatgaatt ttttcaagac cgagattaca 3961 ctggccaatg gagagattcg gaagcgacca cttatcgaaa caaacggaga aacaggagaa 4021 atcgtgtggg acaagggtag ggatttcgcg acagtccgga aggtcctgtc catgccgcag 4081 gtgaacatcg ttaaaaagac cgaagtacag accggaggct tctccaagga aagtatcctc 4141 ccgaaaagga acagcgacaa gctgatcgca cgcaaaaaag attgggaccc caagaaatac 4201 ggcggattcg attctcctac agtcgcttac agtgtactgg ttgtggccaa agtggagaaa 4261 gggaagtcta aaaaactcaa aagcgtcaag gaactgctgg gcatcacaat catggagcga 4321 tcaagcttcg aaaaaaaccc catcgacttt ctcgaggcga aaggatataa agaggtcaaa 4381 aaagacctca tcattaagct tcccaagtac tctctctttg agcttgaaaa cggccggaaa 4441 cgaatgctcg ctagtgcggg cgagctgcag aaaggtaacg agctggcact gccctctaaa 4501 tacgttaatt tcttgtatct ggccagccac tatgaaaagc tcaaagggtc tcccgaagat 4561 aatgagcaga agcagctgtt cgtggaacaa cacaaacact accttgatga gatcatcgag 4621 caaataagcg aattctccaa aagagtgatc ctcgccgacg ctaacctcga taaggtgctt 4681 tctgcttaca ataagcacag ggataagccc atcagggagc aggcagaaaa cattatccac 4741 ttgtttactc tgaccaactt gggcgcgcct gcagccttca agtacttcga caccaccata 4801 gacagaaagc ggtacacctc tacaaaggag gtcctggacg ccacactgat tcatcagtca 4861 attacggggc tctatgaaac aagaatcgac ctctctcagc tcggtggaga cagcagggct 4921 gaccccaaga agaagaggaa ggtgagtggt ggaggaagtg gcgggtcagg gtcggaggcc 4981 agcggttccg gacgggctga cgcattggac gattttgatc tggatatgct gggaagtgac 5041 gccctcgatg attttgacct tgacatgctt ggttcggatg cccttgatga ctttgacctc 5101 gacatgctcg gcagtgacgc ccttgatgat ttcgacctgg acatgctgat taactctaga 5161 agttccggat ctccgaaaaa gaaacgcaaa gttggttcgg gaggtggttc gggtggctct 5221 ggatcagtgc tgcctcaggc tcctgctcct gcaccagctc cagccatggt gtctgcactg 5281 gctcaggcac cagcacccgt gcctgtgctg gctcctggac ctccacaggc tgtggctcca 5341 ccagccccta aacctacaca ggccggcgag ggcacactgt ctgaagctct gctgcagctg 5401 cagttcgacg acgaggatct gggagccctg ctgggaaaca gcaccgatcc tgccgtgttc 5461 accgacctgg ccagcgtgga caacagcgag ttccagcagc tgctgaacca gggcatccct 5521 gtggcccctc acaccaccga gcccatgctg atggaatacc ccgaggccat cacccggctc 5581 gtgacaggcg ctcagaggcc tcctgatcca gctcctgccc ctctgggagc accaggcctg 5641 cctaatggac tgctgtctgg cgacgaggac ttcagctcta tcgccgatat ggatttctca 5701 gccttgctgt caggcggtgg tagtggtggg agcggtagtg acctttccca tccgccccca 5761 aggggccatc tggatgagct gacaaccaca cttgagtcca tgaccgagga tctgaacctg 5821 gactcacccc tgaccccgga attgaacgag attctggata ccttcctgaa cgacgagtgc 5881 ctcttgcatg ccatgcatat cagcacagga ctgtccatct tcgacacatc tctgttttag 5941 aagaattcga tccctaccgg ttagtaatga gtttaaacgg gggaggctaa ctgaaacacg 6001 gaaggagaca ataccggaag gaacccgcgc tatgacggca ataaaaagac agaataaaac 6061 gcacgggtgt tgggtcgttt gttcataaac gcggggttcg gtcccagggc tggcactctg 6121 tcgatacccc accgagaccc cattggggcc aatacgcccg cgtttcttcc ttttccccac 6181 cccacccccc aagttcgggt gaaggcccag ggctcgcagc caacgtcggg gcggcaggcc 6241 ctccatagtc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac 6301 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa 6361 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg 6421 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa 6481 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc 6541 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac 6601 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac 6661 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg 6721 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt 6781 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa 6841 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct 6901 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga 6961 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg 7021 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct 7081 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt 7141 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc 7201 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg 7261 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag 7321 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt 7381 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag 7441 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt 7501 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca 7561 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg 7621 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat 7681 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta 7741 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca 7801 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct 7861 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat 7921 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa 7981 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt 8041 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa 8101 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtcgacggat 8161 cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg atgccgcata 8221 gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt gcgcgagc //