LOCUS       sequence-67416-e        4365 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Cas9m2 Activator.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 4365)
  AUTHORS   Mali P, Aach J, Stranges PB, Esvelt KM, Moosburner M, Kosuri S, Yang
            L, Church GM
  TITLE     CAS9 transcriptional activators for target specificity screening and
            paired nickases for cooperative genome engineering.
  JOURNAL   Nat Biotechnol. 2013 Aug 1. doi: 10.1038/nbt.2675.
  PUBMED    23907171
REFERENCE   2  (bases 1 to 4365)
  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: "Nat 
            Biotechnol. 2013 Aug 1. doi: 10.1038/nbt.2675."
COMMENT     Sequence Label: Cas9m2-VP64
FEATURES             Location/Qualifiers
     source          1..4365
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     CDS             10..30
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(40..66,67..69,70..2553,2554..2559,2560..2625,
                     2626..2628,2629..4143)
                     /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(40..66,67..69,70..2556,2557..2559,2560..4143)
                     /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             40..4143
                     /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             4156..4176
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4201..4350
                     /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 gccaccatgc ccaagaagaa gaggaaggtg ggaaggggga tggacaagaa gtactccatt
       61 gggctcgcta tcggcacaaa cagcgtcggc tgggccgtca ttacggacga gtacaaggtg
      121 ccgagcaaaa aattcaaagt tctgggcaat accgatcgcc acagcataaa gaagaacctc
      181 attggcgccc tcctgttcga ctccggggag acggccgaag ccacgcggct caaaagaaca
      241 gcacggcgca gatatacccg cagaaagaat cggatctgct acctgcagga gatctttagt
      301 aatgagatgg ctaaggtgga tgactctttc ttccataggc tggaggagtc ctttttggtg
      361 gaggaggata aaaagcacga gcgccaccca atctttggca atatcgtgga cgaggtggcg
      421 taccatgaaa agtacccaac catatatcat ctgaggaaga agcttgtaga cagtactgat
      481 aaggctgact tgcggttgat ctatctcgcg ctggcgcata tgatcaaatt tcggggacac
      541 ttcctcatcg agggggacct gaacccagac aacagcgatg tcgacaaact ctttatccaa
      601 ctggttcaga cttacaatca gcttttcgaa gagaacccga tcaacgcatc cggagttgac
      661 gccaaagcaa tcctgagcgc taggctgtcc aaatcccggc ggctcgaaaa cctcatcgca
      721 cagctccctg gggagaagaa gaacggcctg tttggtaatc ttatcgccct gtcactcggg
      781 ctgaccccca actttaaatc taacttcgac ctggccgaag atgccaagct tcaactgagc
      841 aaagacacct acgatgatga tctcgacaat ctgctggccc agatcggcga ccagtacgca
      901 gacctttttt tggcggcaaa gaacctgtca gacgccattc tgctgagtga tattctgcga
      961 gtgaacacgg agatcaccaa agctccgctg agcgctagta tgatcaagcg ctatgatgag
     1021 caccaccaag acttgacttt gctgaaggcc cttgtcagac agcaactgcc tgagaagtac
     1081 aaggaaattt tcttcgatca gtctaaaaat ggctacgccg gatacattga cggcggagca
     1141 agccaggagg aattttacaa atttattaag cccatcttgg aaaaaatgga cggcaccgag
     1201 gagctgctgg taaagcttaa cagagaagat ctgttgcgca aacagcgcac tttcgacaat
     1261 ggaagcatcc cccaccagat tcacctgggc gaactgcacg ctatcctcag gcggcaagag
     1321 gatttctacc cctttttgaa agataacagg gaaaagattg agaaaatcct cacatttcgg
     1381 ataccctact atgtaggccc cctcgcccgg ggaaattcca gattcgcgtg gatgactcgc
     1441 aaatcagaag agaccatcac tccctggaac ttcgaggaag tcgtggataa gggggcctct
     1501 gcccagtcct tcatcgaaag gatgactaac tttgataaaa atctgcctaa cgaaaaggtg
     1561 cttcctaaac actctctgct gtacgagtac ttcacagttt ataacgagct caccaaggtc
     1621 aaatacgtca cagaagggat gagaaagcca gcattcctgt ctggagagca gaagaaagct
     1681 atcgtggacc tcctcttcaa gacgaaccgg aaagttaccg tgaaacagct caaagaagac
     1741 tatttcaaaa agattgaatg tttcgactct gttgaaatca gcggagtgga ggatcgcttc
     1801 aacgcatccc tgggaacgta tcacgatctc ctgaaaatca ttaaagacaa ggacttcctg
     1861 gacaatgagg agaacgagga cattcttgag gacattgtcc tcacccttac gttgtttgaa
     1921 gatagggaga tgattgaaga acgcttgaaa acttacgctc atctcttcga cgacaaagtc
     1981 atgaaacagc tcaagaggcg ccgatataca ggatgggggc ggctgtcaag aaaactgatc
     2041 aatgggatcc gagacaagca gagtggaaag acaatcctgg attttcttaa gtccgatgga
     2101 tttgccaacc ggaacttcat gcagttgatc catgatgact ctctcacctt taaggaggac
     2161 atccagaaag cacaagtttc tggccagggg gacagtcttc acgagcacat cgctaatctt
     2221 gcaggtagcc cagctatcaa aaagggaata ctgcagaccg ttaaggtcgt ggatgaactc
     2281 gtcaaagtaa tgggaaggca taagcccgag aatatcgtta tcgagatggc ccgagagaac
     2341 caaactaccc agaagggaca gaagaacagt agggaaagga tgaagaggat tgaagagggt
     2401 ataaaagaac tggggtccca aatccttaag gaacacccag ttgaaaacac ccagcttcag
     2461 aatgagaagc tctacctgta ctacctgcag aacggcaggg acatgtacgt ggatcaggaa
     2521 ctggacatca atcggctctc cgactacgac gtggatgcta tcgtgcccca gtcttttctc
     2581 aaagatgatt ctattgataa taaagtgttg acaagatccg ataaaaatag agggaagagt
     2641 gataacgtcc cctcagaaga agttgtcaag aaaatgaaaa attattggcg gcagctgctg
     2701 aacgccaaac tgatcacaca acggaagttc gataatctga ctaaggctga acgaggtggc
     2761 ctgtctgagt tggataaagc cggcttcatc aaaaggcagc ttgttgagac acgccagatc
     2821 accaagcacg tggcccaaat tctcgattca cgcatgaaca ccaagtacga tgaaaatgac
     2881 aaactgattc gagaggtgaa agttattact ctgaagtcta agctggtctc agatttcaga
     2941 aaggactttc agttttataa ggtgagagag atcaacaatt accaccatgc gcatgatgcc
     3001 tacctgaatg cagtggtagg cactgcactt atcaaaaaat atcccaagct tgaatctgaa
     3061 tttgtttacg gagactataa agtgtacgat gttaggaaaa tgatcgcaaa gtctgagcag
     3121 gaaataggca aggccaccgc taagtacttc ttttacagca atattatgaa ttttttcaag
     3181 accgagatta cactggccaa tggagagatt cggaagcgac cacttatcga aacaaacgga
     3241 gaaacaggag aaatcgtgtg ggacaagggt agggatttcg cgacagtccg gaaggtcctg
     3301 tccatgccgc aggtgaacat cgttaaaaag accgaagtac agaccggagg cttctccaag
     3361 gaaagtatcc tcccgaaaag gaacagcgac aagctgatcg cacgcaaaaa agattgggac
     3421 cccaagaaat acggcggatt cgattctcct acagtcgctt acagtgtact ggttgtggcc
     3481 aaagtggaga aagggaagtc taaaaaactc aaaagcgtca aggaactgct gggcatcaca
     3541 atcatggagc gatcaagctt cgaaaaaaac cccatcgact ttctcgaggc gaaaggatat
     3601 aaagaggtca aaaaagacct catcattaag cttcccaagt actctctctt tgagcttgaa
     3661 aacggccgga aacgaatgct cgctagtgcg ggcgagctgc agaaaggtaa cgagctggca
     3721 ctgccctcta aatacgttaa tttcttgtat ctggccagcc actatgaaaa gctcaaaggg
     3781 tctcccgaag ataatgagca gaagcagctg ttcgtggaac aacacaaaca ctaccttgat
     3841 gagatcatcg agcaaataag cgaattctcc aaaagagtga tcctcgccga cgctaacctc
     3901 gataaggtgc tttctgctta caataagcac agggataagc ccatcaggga gcaggcagaa
     3961 aacattatcc acttgtttac tctgaccaac ttgggcgcgc ctgcagcctt caagtacttc
     4021 gacaccacca tagacagaaa gcggtacacc tctacaaagg aggtcctgga cgccacactg
     4081 attcatcagt caattacggg gctctatgaa acaagaatcg acctctctca gctcggtgga
     4141 gacagcaggg ctgaccccaa gaagaagagg aaggtggagg ccagcggttc cggacgggct
     4201 gacgcattgg acgattttga tctggatatg ctgggaagtg acgccctcga tgattttgac
     4261 cttgacatgc ttggttcgga tgcccttgat gactttgacc tcgacatgct cggcagtgac
     4321 gcccttgatg atttcgacct ggacatgctg attaactcta gatga
//
