LOCUS       sequence-78959-e        4486 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Expresses Cas9-nickase in mammalian cells and zygotes..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 4486)
  AUTHORS   Fujii W, Onuma A, Sugiura K, Naito K
  TITLE     Efficient generation of genome-modified mice via offset-nicking by 
            CRISPR/Cas system.
  JOURNAL   Biochem Biophys Res Commun. 2014 Jan 31. pii: S0006-291X(14)00176-4.
            doi: 10.1016/j.bbrc.2014.01.141.
  PUBMED    24491566
REFERENCE   2  (bases 1 to 4486)
  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: "Biochem 
            Biophys Res Commun. 2014 Jan 31. pii: S0006-291X(14)00176-4. doi: 
            10.1016/j.bbrc.2014.01.141."
COMMENT     Sequence Label: pCAG-T3-hCasD10A-pA
FEATURES             Location/Qualifiers
     source          1..4486
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     promoter        1..19
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     CDS             80..103
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             113..133
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(158..181,182..184,185..4258)
                     /codon_start=1
                     /product="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
                     /label=Cas9(D10A)
                     /note="generates RNA-guided single strand nicks in DNA due 
                     to the D10A mutation in the RuvC catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             158..4258
                     /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="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             4271..4291
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
ORIGIN
        1 aattaaccct cactaaaggg aacaaaagct ggagctccac cgcggtggcg gccgctctag
       61 cccgggcgga tccaccatgg attacaagga tgacgacgat aagatcatgg ccccaaagaa
      121 gaagcggaag gtcggtatcc acggagtccc agcagccgac aagaagtact ccattgggct
      181 cgccatcggc acaaacagcg tcggctgggc cgtcattacg gacgagtaca aggtgccgag
      241 caaaaaattc aaagttctgg gcaataccga tcgccacagc ataaagaaga acctcattgg
      301 cgccctcctg ttcgactccg gggagacggc cgaagccacg cggctcaaaa gaacagcacg
      361 gcgcagatat acccgcagaa agaatcggat ctgctacctg caggagatct ttagtaatga
      421 gatggctaag gtggatgact ctttcttcca taggctggag gagtcctttt tggtggagga
      481 ggataaaaag cacgagcgcc acccaatctt tggcaatatc gtggacgagg tggcgtacca
      541 tgaaaagtac ccaaccatat atcatctgag gaagaagctt gtagacagta ctgataaggc
      601 tgacttgcgg ttgatctatc tcgcgctggc gcatatgatc aaatttcggg gacacttcct
      661 catcgagggg gacctgaacc cagacaacag cgatgtcgac aaactcttta tccaactggt
      721 tcagacttac aatcagcttt tcgaagagaa cccgatcaac gcatccggag ttgacgccaa
      781 agcaatcctg agcgctaggc tgtccaaatc ccggcggctc gaaaacctca tcgcacagct
      841 ccctggggag aagaagaacg gcctgtttgg taatcttatc gccctgtcac tcgggctgac
      901 ccccaacttt aaatctaact tcgacctggc cgaagatgcc aagcttcaac tgagcaaaga
      961 cacctacgat gatgatctcg acaatctgct ggcccagatc ggcgaccagt acgcagacct
     1021 ttttttggcg gcaaagaacc tgtcagacgc cattctgctg agtgatattc tgcgagtgaa
     1081 cacggagatc accaaagctc cgctgagcgc tagtatgatc aagcgctatg atgagcacca
     1141 ccaagacttg actttgctga aggcccttgt cagacagcaa ctgcctgaga agtacaagga
     1201 aattttcttc gatcagtcta aaaatggcta cgccggatac attgacggcg gagcaagcca
     1261 ggaggaattt tacaaattta ttaagcccat cttggaaaaa atggacggca ccgaggagct
     1321 gctggtaaag cttaacagag aagatctgtt gcgcaaacag cgcactttcg acaatggaag
     1381 catcccccac cagattcacc tgggcgaact gcacgctatc ctcaggcggc aagaggattt
     1441 ctaccccttt ttgaaagata acagggaaaa gattgagaaa atcctcacat ttcggatacc
     1501 ctactatgta ggccccctcg cccggggaaa ttccagattc gcgtggatga ctcgcaaatc
     1561 agaagagacc atcactccct ggaacttcga ggaagtcgtg gataaggggg cctctgccca
     1621 gtccttcatc gaaaggatga ctaactttga taaaaatctg cctaacgaaa aggtgcttcc
     1681 taaacactct ctgctgtacg agtacttcac agtttataac gagctcacca aggtcaaata
     1741 cgtcacagaa gggatgagaa agccagcatt cctgtctgga gagcagaaga aagctatcgt
     1801 ggacctcctc ttcaagacga accggaaagt taccgtgaaa cagctcaaag aagactattt
     1861 caaaaagatt gaatgtttcg actctgttga aatcagcgga gtggaggatc gcttcaacgc
     1921 atccctggga acgtatcacg atctcctgaa aatcattaaa gacaaggact tcctggacaa
     1981 tgaggagaac gaggacattc ttgaggacat tgtcctcacc cttacgttgt ttgaagatag
     2041 ggagatgatt gaagaacgct tgaaaactta cgctcatctc ttcgacgaca aagtcatgaa
     2101 acagctcaag aggcgccgat atacaggatg ggggcggctg tcaagaaaac tgatcaatgg
     2161 gatccgagac aagcagagtg gaaagacaat cctggatttt cttaagtccg atggatttgc
     2221 caaccggaac ttcatgcagt tgatccatga tgactctctc acctttaagg aggacatcca
     2281 gaaagcacaa gtttctggcc agggggacag tcttcacgag cacatcgcta atcttgcagg
     2341 tagcccagct atcaaaaagg gaatactgca gaccgttaag gtcgtggatg aactcgtcaa
     2401 agtaatggga aggcataagc ccgagaatat cgttatcgag atggcccgag agaaccaaac
     2461 tacccagaag ggacagaaga acagtaggga aaggatgaag aggattgaag agggtataaa
     2521 agaactgggg tcccaaatcc ttaaggaaca cccagttgaa aacacccagc ttcagaatga
     2581 gaagctctac ctgtactacc tgcagaacgg cagggacatg tacgtggatc aggaactgga
     2641 catcaatcgg ctctccgact acgacgtgga tcatatcgtg ccccagtctt ttctcaaaga
     2701 tgattctatt gataataaag tgttgacaag atccgataaa aatagaggga agagtgataa
     2761 cgtcccctca gaagaagttg tcaagaaaat gaaaaattat tggcggcagc tgctgaacgc
     2821 caaactgatc acacaacgga agttcgataa tctgactaag gctgaacgag gtggcctgtc
     2881 tgagttggat aaagccggct tcatcaaaag gcagcttgtt gagacacgcc agatcaccaa
     2941 gcacgtggcc caaattctcg attcacgcat gaacaccaag tacgatgaaa atgacaaact
     3001 gattcgagag gtgaaagtta ttactctgaa gtctaagctg gtctcagatt tcagaaagga
     3061 ctttcagttt tataaggtga gagagatcaa caattaccac catgcgcatg atgcctacct
     3121 gaatgcagtg gtaggcactg cacttatcaa aaaatatccc aagcttgaat ctgaatttgt
     3181 ttacggagac tataaagtgt acgatgttag gaaaatgatc gcaaagtctg agcaggaaat
     3241 aggcaaggcc accgctaagt acttctttta cagcaatatt atgaattttt tcaagaccga
     3301 gattacactg gccaatggag agattcggaa gcgaccactt atcgaaacaa acggagaaac
     3361 aggagaaatc gtgtgggaca agggtaggga tttcgcgaca gtccggaagg tcctgtccat
     3421 gccgcaggtg aacatcgtta aaaagaccga agtacagacc ggaggcttct ccaaggaaag
     3481 tatcctcccg aaaaggaaca gcgacaagct gatcgcacgc aaaaaagatt gggaccccaa
     3541 gaaatacggc ggattcgatt ctcctacagt cgcttacagt gtactggttg tggccaaagt
     3601 ggagaaaggg aagtctaaaa aactcaaaag cgtcaaggaa ctgctgggca tcacaatcat
     3661 ggagcgatca agcttcgaaa aaaaccccat cgactttctc gaggcgaaag gatataaaga
     3721 ggtcaaaaaa gacctcatca ttaagcttcc caagtactct ctctttgagc ttgaaaacgg
     3781 ccggaaacga atgctcgcta gtgcgggcga gctgcagaaa ggtaacgagc tggcactgcc
     3841 ctctaaatac gttaatttct tgtatctggc cagccactat gaaaagctca aagggtctcc
     3901 cgaagataat gagcagaagc agctgttcgt ggaacaacac aaacactacc ttgatgagat
     3961 catcgagcaa ataagcgaat tctccaaaag agtgatcctc gccgacgcta acctcgataa
     4021 ggtgctttct gcttacaata agcacaggga taagcccatc agggagcagg cagaaaacat
     4081 tatccacttg tttactctga ccaacttggg cgcgcctgca gccttcaagt acttcgacac
     4141 caccatagac agaaagcggt acacctctac aaaggaggtc ctggacgcca cactgattca
     4201 tcagtcaatt acggggctct atgaaacaag aatcgacctc tctcagctcg gtggagacag
     4261 cagggctgac cccaagaaga agaggaaggt gtgaatcgat ggtacctatg ggcaccaaag
     4321 aacctgtaaa cgttatcttt ttaaattgaa tgtgcacaaa taaaagtttg gaaaagaaaa
     4381 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
     4441 aaaaaaaaaa aaaaaaaaaa aaaaaaaaac cctcgaggta gcatgc
//
