LOCUS Exported 7653 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Mammalian vector for expressing effector domains fused to dCas9.. ACCESSION . VERSION . KEYWORDS pdCas9-NED SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7653) AUTHORS Goubert D, Koncz M, Kiss A, Rots MG TITLE Establishment of Cell Lines Stably Expressing dCas9-Fusions to Address Kinetics of Epigenetic Editing. JOURNAL Methods Mol Biol. 2018;1767:395-415. doi: 10.1007/978-1-4939-7774-1_22. PUBMED 29524148 REFERENCE 2 (bases 1 to 7653) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7653 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 132..335 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 286..306 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 377..396 /label=T7 /note="T7 promoter, forward primer" promoter 377..395 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" regulatory 403..412 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 409..4512 /codon_start=1 /product="catalytically dead mutant of the Cas9 endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /label=dCas9 /note="RNA-guided DNA-binding protein that lacks endonuclease activity due to the D10A mutation in the RuvC catalytic domain and the H840A mutation in the HNH catalytic domain" /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL DATLIHQSITGLYETRIDLSQLGGD" CDS 4519..4539 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 4546..4611 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 4668..4685 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(4708..4725) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 4714..4938 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(5009..5025) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5009..5025) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(5022..5044) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 5033..5049 /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(5057..5087) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5102..5123 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(5240..5257) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5411..5999) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5491..5510) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6170..7030) /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 6793..6812 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7031..7135) /gene="bla" /label=AmpR promoter primer_bind complement(7214..7233) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 7405..131 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" ORIGIN 1 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg 61 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg 121 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact 181 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa 241 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta 301 ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct 361 agagatccgc ggccgctaat acgactcact atagggagag ccgccaccat ggataaaaag 421 tattctattg gtttagccat cggcactaat tccgttggat gggctgtcat aaccgatgaa 481 tacaaagtac cttcaaagaa atttaaggtg ttggggaaca cagaccgtca ttcgattaaa 541 aagaatctta tcggtgccct cctattcgat agtggcgaaa cggcagaggc gactcgcctg 601 aaacgaaccg ctcggagaag gtatacacgt cgcaagaacc gaatatgtta cttacaagaa 661 atttttagca atgagatggc caaagttgac gattctttct ttcaccgttt ggaagagtcc 721 ttccttgtcg aagaggacaa gaaacatgaa cggcacccca tctttggaaa catagtagat 781 gaggtggcat atcatgaaaa gtacccaacg atttatcacc tcagaaaaaa gctagttgac 841 tcaactgata aagcggacct gaggttaatc tacttggctc ttgcccatat gataaagttc 901 cgtgggcact ttctcattga gggtgatcta aatccggaca actcggatgt cgacaaactg 961 ttcatccagt tagtacaaac ctataatcag ttgtttgaag agaaccctat aaatgcaagt 1021 ggcgtggatg cgaaggctat tcttagcgcc cgcctctcta aatcccgacg gctagaaaac 1081 ctgatcgcac aattacccgg agagaagaaa aatgggttgt tcggtaacct tatagcgctc 1141 tcactaggcc tgacaccaaa ttttaagtcg aacttcgact tagctgaaga tgccaaattg 1201 cagcttagta aggacacgta cgatgacgat ctcgacaatc tactggcaca aattggagat 1261 cagtatgcgg acttattttt ggctgccaaa aaccttagcg atgcaatcct cctatctgac 1321 atactgagag ttaatactga gattaccaag gcgccgttat ccgcttcaat gatcaaaagg 1381 tacgatgaac atcaccaaga cttgacactt ctcaaggccc tagtccgtca gcaactgcct 1441 gagaaatata aggaaatatt ctttgatcag tcgaaaaacg ggtacgcagg ttatattgac 1501 ggcggagcga gtcaagagga attctacaag tttatcaaac ccatattaga gaagatggat 1561 gggacggaag agttgcttgt aaaactcaat cgcgaagatc tactgcgaaa gcagcggact 1621 ttcgacaacg gtagcattcc acatcaaatc cacttaggcg aattgcatgc tatacttaga 1681 aggcaggagg atttttatcc gttcctcaaa gacaatcgtg aaaagattga gaaaatccta 1741 acctttcgca taccttacta tgtgggaccc ctggcccgag ggaactctcg gttcgcatgg 1801 atgacaagaa agtccgaaga aacgattact ccatggaatt ttgaggaagt tgtcgataaa 1861 ggtgcgtcag ctcaatcgtt catcgagagg atgaccaact ttgacaagaa tttaccgaac 1921 gaaaaagtat tgcctaagca cagtttactt tacgagtatt tcacagtgta caatgaactc 1981 acgaaagtta agtatgtcac tgagggcatg cgtaaacccg cctttctaag cggagaacag 2041 aagaaagcaa tagtagatct gttattcaag accaaccgca aagtgacagt taagcaattg 2101 aaagaggact actttaagaa aattgaatgc ttcgattctg tcgagatctc cggggtagaa 2161 gatcgattta atgcgtcact tggtacgtat catgacctcc taaagataat taaagataag 2221 gacttcctgg ataacgaaga gaatgaagat atcttagaag atatagtgtt gactcttacc 2281 ctctttgaag atcgggaaat gattgaggaa agactaaaaa catacgctca cctgttcgac 2341 gataaggtta tgaaacagtt aaagaggcgt cgctatacgg gctggggacg attgtcgcgg 2401 aaacttatca acgggataag agacaagcaa agtggtaaaa ctattctcga ttttctaaag 2461 agcgacggct tcgccaatag gaactttatg cagctgatcc atgatgactc tttaaccttc 2521 aaagaggata tacaaaaggc acaggtttcc ggacaagggg actcattgca cgaacatatt 2581 gcgaatcttg ctggttcgcc agccatcaaa aagggcatac tccagacagt caaagtagtg 2641 gatgagctag ttaaggtcat gggacgtcac aaaccggaaa acattgtaat cgagatggca 2701 cgcgaaaatc aaacgactca gaaggggcaa aaaaacagtc gagagcggat gaagagaata 2761 gaagagggta ttaaagaact gggcagccag atcttaaagg agcatcctgt ggaaaatacc 2821 caattgcaga acgagaaact ttacctctat tacctacaaa atggaaggga catgtatgtt 2881 gatcaggaac tggacataaa ccgtttatct gattacgacg tcgatgccat tgtaccccaa 2941 tcctttttga aggacgattc aatcgacaat aaagtgctta cacgctcgga taagaaccga 3001 gggaaaagtg acaatgttcc aagcgaggaa gtcgtaaaga aaatgaagaa ctattggcgg 3061 cagctcctaa atgcgaaact gataacgcaa agaaagttcg ataacttaac taaagctgag 3121 aggggtggct tgtctgaact tgacaaggcc ggatttatta aacgtcagct cgtggaaacc 3181 cgccaaatca caaagcatgt tgcacagata ctagattccc gaatgaatac gaaatacgac 3241 gagaacgata agctgattcg ggaagtcaaa gtaatcactt taaagtcaaa attggtgtcg 3301 gacttcagaa aggattttca attctataaa gttagggaga taaataacta ccaccatgcg 3361 cacgacgctt atcttaatgc cgtcgtaggg accgcactca ttaagaaata cccgaagcta 3421 gaaagtgagt ttgtgtatgg tgattacaaa gtttatgacg tccgtaagat gatcgcgaaa 3481 agcgaacagg agataggcaa ggctacagcc aaatacttct tttattctaa cattatgaat 3541 ttctttaaga cggaaatcac tctggcaaac ggagagatac gcaaacgacc tttaattgaa 3601 accaatgggg agacaggtga aatcgtatgg gataagggcc gggacttcgc gacggtgaga 3661 aaagttttgt ccatgcccca agtcaacata gtaaagaaaa ctgaggtgca gaccggaggg 3721 ttttcaaagg aatcgattct tccaaaaagg aatagtgata agctcatcgc tcgtaaaaag 3781 gactgggacc cgaaaaagta cggtggcttc gatagcccta cagttgccta ttctgtccta 3841 gtagtggcaa aagttgagaa gggaaaatcc aagaaactga agtcagtcaa agaattattg 3901 gggataacga ttatggagcg ctcgtctttt gaaaagaacc ccatcgactt ccttgaggcg 3961 aaaggttaca aggaagtaaa aaaggatctc ataattaaac taccaaagta tagtctgttt 4021 gagttagaaa atggccgaaa acggatgttg gctagcgccg gagagcttca aaaggggaac 4081 gaactcgcac taccgtctaa atacgtgaat ttcctgtatt tagcgtccca ttacgagaag 4141 ttgaaaggtt cacctgaaga taacgaacag aagcaacttt ttgttgagca gcacaaacat 4201 tatctcgacg aaatcataga gcaaatttcg gaattcagta agagagtcat cctagctgat 4261 gccaatctgg acaaagtatt aagcgcatac aacaagcaca gggataaacc catacgtgag 4321 caggcggaaa atattatcca tttgtttact cttaccaacc tcggcgctcc agccgcattc 4381 aagtattttg acacaacgat agatcgcaaa cgatacactt ctaccaagga ggtgctagac 4441 gcgacactga ttcaccaatc catcacggga ttatatgaaa ctcggataga tttgtcacag 4501 cttgggggtg acggatcccc caagaagaag aggaaagtct cgagcgacta caaagaccat 4561 gacggtgatt ataaagatca tgacatcgat tacaaggatg acgatgacaa ggctgcagga 4621 ggcggtggaa gcgggcgcgc ccatatgtta attaacaatt aaccggtcat catcaccatc 4681 accattgagt ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg 4741 ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt 4801 cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg 4861 gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg 4921 atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctcg ataccgtcga 4981 cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc 5041 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tagggtgcct 5101 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa 5161 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta 5221 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc 5281 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg 5341 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt 5401 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa 5461 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct 5521 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc 5581 cttcgggaag cgtggcgctt tctcaatgct cacgctgtag gtatctcagt tcggtgtagg 5641 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct 5701 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag 5761 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga 5821 agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga 5881 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg 5941 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag 6001 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag 6061 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat 6121 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct 6181 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac 6241 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa 6301 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg 6361 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt 6421 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca 6481 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt 6541 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct 6601 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg 6661 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg 6721 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg 6781 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa 6841 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt 6901 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt 6961 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt 7021 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca 7081 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat 7141 ttccccgaaa agtgccacct gacgtcgacg gatcgggaga tcgatctccc gatcccctag 7201 ggtcgactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat ctgctccctg 7261 cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag 7321 gcttgaccga caattgcatg aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg 7381 atgtacgggc cagatatacg cgttgacatt gattattgac tagttattaa tagtaatcaa 7441 ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa 7501 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg 7561 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggac tatttacggt 7621 aaactgccca cttggcagta catcaagtgt atc //