LOCUS       sequence-71934-e        4410 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  encodes human-optimized dCas9_VP64 synthetic transcription factor.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 4410)
  AUTHORS   Farzadfard F, Perli SD, Lu TK
  TITLE     Tunable and Multifunctional Eukaryotic Transcription Factors Based 
            on CRISPR/Cas.
  JOURNAL   ACS Synth Biol. 2013 Sep 11.
  PUBMED    23977949
REFERENCE   2  (bases 1 to 4410)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "ACS Synth 
            Biol. 2013 Sep 11."
COMMENT     Sequence Label: pCMV_dCas9_VP64
FEATURES             Location/Qualifiers
     source          1..4410
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     CDS             join(1..27,28..30,31..2514,2515..2520,2521..2586,2587..2589,
                     2590..4104)
                     /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
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             join(1..27,28..30,31..2517,2518..2520,2521..4104)
                     /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             1..4104
                     /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="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             4141..4161
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4168..4188
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4195..4215
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4255..4404
                     /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"
ORIGIN
        1 atggacaaga agtactccat tgggctcgcc atcggcacaa acagcgtcgg ctgggccgtc
       61 attacggacg agtacaaggt gccgagcaaa aaattcaaag ttctgggcaa taccgatcgc
      121 cacagcataa agaagaacct cattggcgcc ctcctgttcg actccgggga gacggccgaa
      181 gccacgcggc tcaaaagaac agcacggcgc agatataccc gcagaaagaa tcggatctgc
      241 tacctgcagg agatctttag taatgagatg gctaaggtgg atgactcttt cttccatagg
      301 ctggaggagt cctttttggt ggaggaggat aaaaagcacg agcgccaccc aatctttggc
      361 aatatcgtgg acgaggtggc gtaccatgaa aagtacccaa ccatatatca tctgaggaag
      421 aagcttgtag acagtactga taaggctgac ttgcggttga tctatctcgc gctggcgcat
      481 atgatcaaat ttcggggaca cttcctcatc gagggggacc tgaacccaga caacagcgat
      541 gtcgacaaac tctttatcca actggttcag acttacaatc agcttttcga agagaacccg
      601 atcaacgcat ccggagttga cgccaaagca atcctgagcg ctaggctgtc caaatcccgg
      661 cggctcgaaa acctcatcgc acagctccct ggggagaaga agaacggcct gtttggtaat
      721 cttatcgccc tgtcactcgg gctgaccccc aactttaaat ctaacttcga cctggccgaa
      781 gatgccaagc ttcaactgag caaagacacc tacgatgatg atctcgacaa tctgctggcc
      841 cagatcggcg accagtacgc agaccttttt ttggcggcaa agaacctgtc agacgccatt
      901 ctgctgagtg atattctgcg agtgaacacg gagatcacca aagctccgct gagcgctagt
      961 atgatcaagc gctatgatga gcaccaccaa gacttgactt tgctgaaggc ccttgtcaga
     1021 cagcaactgc ctgagaagta caaggaaatt ttcttcgatc agtctaaaaa tggctacgcc
     1081 ggatacattg acggcggagc aagccaggag gaattttaca aatttattaa gcccatcttg
     1141 gaaaaaatgg acggcaccga ggagctgctg gtaaagctta acagagaaga tctgttgcgc
     1201 aaacagcgca ctttcgacaa tggaagcatc ccccaccaga ttcacctggg cgaactgcac
     1261 gctatcctca ggcggcaaga ggatttctac ccctttttga aagataacag ggaaaagatt
     1321 gagaaaatcc tcacatttcg gataccctac tatgtaggcc ccctcgcccg gggaaattcc
     1381 agattcgcgt ggatgactcg caaatcagaa gagaccatca ctccctggaa cttcgaggaa
     1441 gtcgtggata agggggcctc tgcccagtcc ttcatcgaaa ggatgactaa ctttgataaa
     1501 aatctgccta acgaaaaggt gcttcctaaa cactctctgc tgtacgagta cttcacagtt
     1561 tataacgagc tcaccaaggt caaatacgtc acagaaggga tgagaaagcc agcattcctg
     1621 tctggagagc agaagaaagc tatcgtggac ctcctcttca agacgaaccg gaaagttacc
     1681 gtgaaacagc tcaaagaaga ctatttcaaa aagattgaat gtttcgactc tgttgaaatc
     1741 agcggagtgg aggatcgctt caacgcatcc ctgggaacgt atcacgatct cctgaaaatc
     1801 attaaagaca aggacttcct ggacaatgag gagaacgagg acattcttga ggacattgtc
     1861 ctcaccctta cgttgtttga agatagggag atgattgaag aacgcttgaa aacttacgct
     1921 catctcttcg acgacaaagt catgaaacag ctcaagaggc gccgatatac aggatggggg
     1981 cggctgtcaa gaaaactgat caatgggatc cgagacaagc agagtggaaa gacaatcctg
     2041 gattttctta agtccgatgg atttgccaac cggaacttca tgcagttgat ccatgatgac
     2101 tctctcacct ttaaggagga catccagaaa gcacaagttt ctggccaggg ggacagtctt
     2161 cacgagcaca tcgctaatct tgcaggtagc ccagctatca aaaagggaat actgcagacc
     2221 gttaaggtcg tggatgaact cgtcaaagta atgggaaggc ataagcccga gaatatcgtt
     2281 atcgagatgg cccgagagaa ccaaactacc cagaagggac agaagaacag tagggaaagg
     2341 atgaagagga ttgaagaggg tataaaagaa ctggggtccc aaatccttaa ggaacaccca
     2401 gttgaaaaca cccagcttca gaatgagaag ctctacctgt actacctgca gaacggcagg
     2461 gacatgtacg tggatcagga actggacatc aatcggctct ccgactacga cgtggatgcc
     2521 atcgtgcccc agtcttttct caaagatgat tctattgata ataaagtgtt gacaagatcc
     2581 gataaaaata gagggaagag tgataacgtc ccctcagaag aagttgtcaa gaaaatgaaa
     2641 aattattggc ggcagctgct gaacgccaaa ctgatcacac aacggaagtt cgataatctg
     2701 actaaggctg aacgaggtgg cctgtctgag ttggataaag ccggcttcat caaaaggcag
     2761 cttgttgaga cacgccagat caccaagcac gtggcccaaa ttctcgattc acgcatgaac
     2821 accaagtacg atgaaaatga caaactgatt cgagaggtga aagttattac tctgaagtct
     2881 aagctggtct cagatttcag aaaggacttt cagttttata aggtgagaga gatcaacaat
     2941 taccaccatg cgcatgatgc ctacctgaat gcagtggtag gcactgcact tatcaaaaaa
     3001 tatcccaagc ttgaatctga atttgtttac ggagactata aagtgtacga tgttaggaaa
     3061 atgatcgcaa agtctgagca ggaaataggc aaggccaccg ctaagtactt cttttacagc
     3121 aatattatga attttttcaa gaccgagatt acactggcca atggagagat tcggaagcga
     3181 ccacttatcg aaacaaacgg agaaacagga gaaatcgtgt gggacaaggg tagggatttc
     3241 gcgacagtcc ggaaggtcct gtccatgccg caggtgaaca tcgttaaaaa gaccgaagta
     3301 cagaccggag gcttctccaa ggaaagtatc ctcccgaaaa ggaacagcga caagctgatc
     3361 gcacgcaaaa aagattggga ccccaagaaa tacggcggat tcgattctcc tacagtcgct
     3421 tacagtgtac tggttgtggc caaagtggag aaagggaagt ctaaaaaact caaaagcgtc
     3481 aaggaactgc tgggcatcac aatcatggag cgatcaagct tcgaaaaaaa ccccatcgac
     3541 tttctcgagg cgaaaggata taaagaggtc aaaaaagacc tcatcattaa gcttcccaag
     3601 tactctctct ttgagcttga aaacggccgg aaacgaatgc tcgctagtgc gggcgagctg
     3661 cagaaaggta acgagctggc actgccctct aaatacgtta atttcttgta tctggccagc
     3721 cactatgaaa agctcaaagg gtctcccgaa gataatgagc agaagcagct gttcgtggaa
     3781 caacacaaac actaccttga tgagatcatc gagcaaataa gcgaattctc caaaagagtg
     3841 atcctcgccg acgctaacct cgataaggtg ctttctgctt acaataagca cagggataag
     3901 cccatcaggg agcaggcaga aaacattatc cacttgttta ctctgaccaa cttgggcgcg
     3961 cctgcagcct tcaagtactt cgacaccacc atagacagaa agcggtacac ctctacaaag
     4021 gaggtcctgg acgccacact gattcatcag tcaattacgg ggctctatga aacaagaatc
     4081 gacctctctc agctcggtgg agacagcagg gctgacgggc cctcactggg ttcagggtca
     4141 cccaagaaga aacgcaaagt cgaggatcca aagaagaaaa ggaaggttga agaccccaag
     4201 aaaaagagga aggtggatgg gatcggctca ggcagcaacg gcggtggagg ttcagacgct
     4261 ttggacgatt tcgatctcga tatgctcggt tctgacgccc tggatgattt cgatctggat
     4321 atgctcggca gcgacgctct cgacgatttc gacctcgaca tgctcgggtc agatgccttg
     4381 gatgattttg acctggatat gctctcatga
//
