LOCUS       sequence-67414-e        4146 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Cas9 D10A+H840A.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 4146)
  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 4146)
  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
FEATURES             Location/Qualifiers
     source          1..4146
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     regulatory      1..10
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(7..33,34..36,37..2520,2521..2526,2527..2592,2593..2595,
                     2596..4110)
                     /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(7..33,34..36,37..2523,2524..2526,2527..4110)
                     /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             7..4110
                     /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             4123..4143
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
ORIGIN
        1 gccaccatgg acaagaagta ctccattggg ctcgctatcg gcacaaacag cgtcggctgg
       61 gccgtcatta cggacgagta caaggtgccg agcaaaaaat tcaaagttct gggcaatacc
      121 gatcgccaca gcataaagaa gaacctcatt ggcgccctcc tgttcgactc cggggagacg
      181 gccgaagcca cgcggctcaa aagaacagca cggcgcagat atacccgcag aaagaatcgg
      241 atctgctacc tgcaggagat ctttagtaat gagatggcta aggtggatga ctctttcttc
      301 cataggctgg aggagtcctt tttggtggag gaggataaaa agcacgagcg ccacccaatc
      361 tttggcaata tcgtggacga ggtggcgtac catgaaaagt acccaaccat atatcatctg
      421 aggaagaagc ttgtagacag tactgataag gctgacttgc ggttgatcta tctcgcgctg
      481 gcgcatatga tcaaatttcg gggacacttc ctcatcgagg gggacctgaa cccagacaac
      541 agcgatgtcg acaaactctt tatccaactg gttcagactt acaatcagct tttcgaagag
      601 aacccgatca acgcatccgg agttgacgcc aaagcaatcc tgagcgctag gctgtccaaa
      661 tcccggcggc tcgaaaacct catcgcacag ctccctgggg agaagaagaa cggcctgttt
      721 ggtaatctta tcgccctgtc actcgggctg acccccaact ttaaatctaa cttcgacctg
      781 gccgaagatg ccaagcttca actgagcaaa gacacctacg atgatgatct cgacaatctg
      841 ctggcccaga tcggcgacca gtacgcagac ctttttttgg cggcaaagaa cctgtcagac
      901 gccattctgc tgagtgatat tctgcgagtg aacacggaga tcaccaaagc tccgctgagc
      961 gctagtatga tcaagcgcta tgatgagcac caccaagact tgactttgct gaaggccctt
     1021 gtcagacagc aactgcctga gaagtacaag gaaattttct tcgatcagtc taaaaatggc
     1081 tacgccggat acattgacgg cggagcaagc caggaggaat tttacaaatt tattaagccc
     1141 atcttggaaa aaatggacgg caccgaggag ctgctggtaa agcttaacag agaagatctg
     1201 ttgcgcaaac agcgcacttt cgacaatgga agcatccccc accagattca cctgggcgaa
     1261 ctgcacgcta tcctcaggcg gcaagaggat ttctacccct ttttgaaaga taacagggaa
     1321 aagattgaga aaatcctcac atttcggata ccctactatg taggccccct cgcccgggga
     1381 aattccagat tcgcgtggat gactcgcaaa tcagaagaga ccatcactcc ctggaacttc
     1441 gaggaagtcg tggataaggg ggcctctgcc cagtccttca tcgaaaggat gactaacttt
     1501 gataaaaatc tgcctaacga aaaggtgctt cctaaacact ctctgctgta cgagtacttc
     1561 acagtttata acgagctcac caaggtcaaa tacgtcacag aagggatgag aaagccagca
     1621 ttcctgtctg gagagcagaa gaaagctatc gtggacctcc tcttcaagac gaaccggaaa
     1681 gttaccgtga aacagctcaa agaagactat ttcaaaaaga ttgaatgttt cgactctgtt
     1741 gaaatcagcg gagtggagga tcgcttcaac gcatccctgg gaacgtatca cgatctcctg
     1801 aaaatcatta aagacaagga cttcctggac aatgaggaga acgaggacat tcttgaggac
     1861 attgtcctca cccttacgtt gtttgaagat agggagatga ttgaagaacg cttgaaaact
     1921 tacgctcatc tcttcgacga caaagtcatg aaacagctca agaggcgccg atatacagga
     1981 tgggggcggc tgtcaagaaa actgatcaat gggatccgag acaagcagag tggaaagaca
     2041 atcctggatt ttcttaagtc cgatggattt gccaaccgga acttcatgca gttgatccat
     2101 gatgactctc tcacctttaa ggaggacatc cagaaagcac aagtttctgg ccagggggac
     2161 agtcttcacg agcacatcgc taatcttgca ggtagcccag ctatcaaaaa gggaatactg
     2221 cagaccgtta aggtcgtgga tgaactcgtc aaagtaatgg gaaggcataa gcccgagaat
     2281 atcgttatcg agatggcccg agagaaccaa actacccaga agggacagaa gaacagtagg
     2341 gaaaggatga agaggattga agagggtata aaagaactgg ggtcccaaat ccttaaggaa
     2401 cacccagttg aaaacaccca gcttcagaat gagaagctct acctgtacta cctgcagaac
     2461 ggcagggaca tgtacgtgga tcaggaactg gacatcaatc ggctctccga ctacgacgtg
     2521 gatgctatcg tgccccagtc ttttctcaaa gatgattcta ttgataataa agtgttgaca
     2581 agatccgata aaaatagagg gaagagtgat aacgtcccct cagaagaagt tgtcaagaaa
     2641 atgaaaaatt attggcggca gctgctgaac gccaaactga tcacacaacg gaagttcgat
     2701 aatctgacta aggctgaacg aggtggcctg tctgagttgg ataaagccgg cttcatcaaa
     2761 aggcagcttg ttgagacacg ccagatcacc aagcacgtgg cccaaattct cgattcacgc
     2821 atgaacacca agtacgatga aaatgacaaa ctgattcgag aggtgaaagt tattactctg
     2881 aagtctaagc tggtctcaga tttcagaaag gactttcagt tttataaggt gagagagatc
     2941 aacaattacc accatgcgca tgatgcctac ctgaatgcag tggtaggcac tgcacttatc
     3001 aaaaaatatc ccaagcttga atctgaattt gtttacggag actataaagt gtacgatgtt
     3061 aggaaaatga tcgcaaagtc tgagcaggaa ataggcaagg ccaccgctaa gtacttcttt
     3121 tacagcaata ttatgaattt tttcaagacc gagattacac tggccaatgg agagattcgg
     3181 aagcgaccac ttatcgaaac aaacggagaa acaggagaaa tcgtgtggga caagggtagg
     3241 gatttcgcga cagtccggaa ggtcctgtcc atgccgcagg tgaacatcgt taaaaagacc
     3301 gaagtacaga ccggaggctt ctccaaggaa agtatcctcc cgaaaaggaa cagcgacaag
     3361 ctgatcgcac gcaaaaaaga ttgggacccc aagaaatacg gcggattcga ttctcctaca
     3421 gtcgcttaca gtgtactggt tgtggccaaa gtggagaaag ggaagtctaa aaaactcaaa
     3481 agcgtcaagg aactgctggg catcacaatc atggagcgat caagcttcga aaaaaacccc
     3541 atcgactttc tcgaggcgaa aggatataaa gaggtcaaaa aagacctcat cattaagctt
     3601 cccaagtact ctctctttga gcttgaaaac ggccggaaac gaatgctcgc tagtgcgggc
     3661 gagctgcaga aaggtaacga gctggcactg ccctctaaat acgttaattt cttgtatctg
     3721 gccagccact atgaaaagct caaagggtct cccgaagata atgagcagaa gcagctgttc
     3781 gtggaacaac acaaacacta ccttgatgag atcatcgagc aaataagcga attctccaaa
     3841 agagtgatcc tcgccgacgc taacctcgat aaggtgcttt ctgcttacaa taagcacagg
     3901 gataagccca tcagggagca ggcagaaaac attatccact tgtttactct gaccaacttg
     3961 ggcgcgcctg cagccttcaa gtacttcgac accaccatag acagaaagcg gtacacctct
     4021 acaaaggagg tcctggacgc cacactgatt catcagtcaa ttacggggct ctatgaaaca
     4081 agaatcgacc tctctcagct cggtggagac agcagggctg accccaagaa gaagaggaag
     4141 gtgtga
//
