LOCUS Exported 7540 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Expresses dSp Cas9 fused to truncated RTA activation domain. ACCESSION . VERSION . KEYWORDS dSp-Rta 100-175 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7540) TITLE Church Lab CRISPR plasmids 2017 REFERENCE 2 (bases 1 to 7540) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7540 /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" primer_bind complement(5254..5273) /label=TK-pA-R /note="Thymidine kinase polyA, reverse primer" polyA_signal 5298..5346 /label=HSV TK poly(A) signal /note="herpes simplex virus thymidine kinase polyadenylation signal (Cole and Stacy, 1985)" rep_origin complement(5660..6248) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5740..5759) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6419..7279) /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 7042..7061 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7280..7384) /gene="bla" /label=AmpR promoter primer_bind complement(7459..7478) /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 gtcggtcaaa 4981 gcccttcggg agatggccga tactgtgatt ccccagaagg aagaggctgc aatctgtggc 5041 caaatggacc tttcccatcc gcccccaagg ggccatctgg atgagctgac aaccacactt 5101 gagtccatga ccgaggatct gaacctggac tcacccctga ccccggaatt gaacgagatt 5161 ctggatacct tcctgaacga cgagtgcctc ttgcatgcct agaagaattc gatccctacc 5221 ggttagtaat gagtttaaac gggggaggct aactgaaaca cggaaggaga caataccgga 5281 aggaacccgc gctatgacgg caataaaaag acagaataaa acgcacgggt gttgggtcgt 5341 ttgttcataa acgcggggtt cggtcccagg gctggcactc tgtcgatacc ccaccgagac 5401 cccattgggg ccaatacgcc cgcgtttctt ccttttcccc accccacccc ccaagttcgg 5461 gtgaaggccc agggctcgca gccaacgtcg gggcggcagg ccctccatag tcggtcgttc 5521 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag 5581 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa 5641 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc 5701 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc 5761 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg 5821 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt 5881 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc 5941 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc 6001 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag 6061 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg 6121 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa 6181 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag 6241 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact 6301 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa 6361 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt 6421 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag 6481 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca 6541 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc 6601 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt 6661 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg 6721 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca 6781 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg 6841 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca 6901 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg 6961 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct 7021 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca 7081 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca 7141 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg 7201 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac 7261 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt 7321 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc 7381 cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcgggagat ctcccgatcc 7441 cctatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatctgct 7501 ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc //