LOCUS       sequence-177887-        4215 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Expression plasmid for human codon-optimized dead/inactive SpCas9 
            (without U6-sgRNA coding sequence).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 4215)
  AUTHORS   Kulcsar PI, Talas A, Huszar K, Ligeti Z, Toth E, Weinhardt N, Fodor 
            E, Welker E
  TITLE     Crossing enhanced and high fidelity SpCas9 nucleases to optimize 
            specificity and cleavage.
  JOURNAL   Genome Biol. 2017 Oct 6;18(1):190. doi: 10.1186/s13059-017-1318-8.
  PUBMED    28985763
REFERENCE   2  (bases 1 to 4215)
  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: "Genome 
            Biol."; date: "2017-10-6"; volume: "18(1):190. doi"; pages: " 
            10.1186/s13059-017-1318-8"
COMMENT     Sequence Label: pX330-HA-dSpCas9
FEATURES             Location/Qualifiers
     source          1..4215
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     CDS             4..30
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             34..54
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(64..87,88..90,91..2646,2647..2649,2650..4164)
                     /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 N863A mutation in the HNH 
                     catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             join(64..87,88..90,91..2577,2578..2580,2581..4164)
                     /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="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             4165..4212
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
ORIGIN
        1 atgtacccat acgatgttcc agattacgct tcgccgaaga aaaagcgcaa ggtcgaagcg
       61 tccgacaaga agtacagcat cggcctggcc atcggcacca actctgtggg ctgggccgtg
      121 atcaccgacg agtacaaggt gcccagcaag aaattcaagg tgctgggcaa caccgaccgg
      181 cacagcatca agaagaacct gatcggagcc ctgctgttcg acagcggcga aacagccgag
      241 gccacccggc tgaagagaac cgccagaaga agatacacca gacggaagaa ccggatctgc
      301 tatctgcaag agatcttcag caacgagatg gccaaggtgg acgacagctt cttccacaga
      361 ctggaagagt ccttcctggt ggaagaggat aagaagcacg agcggcaccc catcttcggc
      421 aacatcgtgg acgaggtggc ctaccacgag aagtacccca ccatctacca cctgagaaag
      481 aaactggtgg acagcaccga caaggccgac ctgcggctga tctatctggc cctggcccac
      541 atgatcaagt tccggggcca cttcctgatc gagggcgacc tgaaccccga caacagcgac
      601 gtggacaagc tgttcatcca gctggtgcag acctacaacc agctgttcga ggaaaacccc
      661 atcaacgcca gcggcgtgga cgccaaggcc atcctgtctg ccagactgag caagagcaga
      721 cggctggaaa atctgatcgc ccagctgccc ggcgagaaga agaatggcct gttcggcaac
      781 ctgattgccc tgagcctggg cctgaccccc aacttcaaga gcaacttcga cctggccgag
      841 gatgccaaac tgcagctgag caaggacacc tacgacgacg acctggacaa cctgctggcc
      901 cagatcggcg accagtacgc cgacctgttt ctggccgcca agaacctgtc cgacgccatc
      961 ctgctgagcg acatcctgag agtgaacacc gagatcacca aggcccccct gagcgcctct
     1021 atgatcaaga gatacgacga gcaccaccag gacctgaccc tgctgaaagc tctcgtgcgg
     1081 cagcagctgc ctgagaagta caaagagatt ttcttcgacc agagcaagaa cggctacgcc
     1141 ggctacattg acggcggagc cagccaggaa gagttctaca agttcatcaa gcccatcctg
     1201 gaaaagatgg acggcaccga ggaactgctc gtgaagctga acagagagga cctgctgcgg
     1261 aagcagcgga ccttcgacaa cggcagcatc ccccaccaga tccacctggg agagctgcac
     1321 gccattctgc ggcggcagga agatttttac ccattcctga aggacaaccg ggaaaagatc
     1381 gagaagatcc tgaccttccg catcccctac tacgtgggcc ctctggccag gggaaacagc
     1441 agattcgcct ggatgaccag aaagagcgag gaaaccatca ccccctggaa cttcgaggaa
     1501 gtggtggaca agggcgcttc cgcccagagc ttcatcgagc ggatgaccaa cttcgataag
     1561 aacctgccca acgagaaggt gctgcccaag cacagcctgc tgtacgagta cttcaccgtg
     1621 tataacgagc tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc cgccttcctg
     1681 agcggcgagc agaaaaaggc catcgtggac ctgctgttca agaccaaccg gaaagtgacc
     1741 gtgaagcagc tgaaagagga ctacttcaag aaaatcgagt gcttcgactc cgtggaaatc
     1801 tccggcgtgg aagatcggtt caacgcctcc ctgggcacat accacgatct gctgaaaatt
     1861 atcaaggaca aggacttcct ggacaatgag gaaaacgagg acattctgga agatatcgtg
     1921 ctgaccctga cactgtttga ggacagagag atgatcgagg aacggctgaa aacctatgcc
     1981 cacctgttcg acgacaaagt gatgaagcag ctgaagcggc ggagatacac cggctggggc
     2041 aggctgagcc ggaagctgat caacggcatc cgggacaagc agtccggcaa gacaatcctg
     2101 gatttcctga agtccgacgg cttcgccaac agaaacttca tgcagctgat ccacgacgac
     2161 agcctgacct ttaaagagga catccagaaa gcccaggtgt ccggccaggg cgatagcctg
     2221 cacgagcaca ttgccaatct ggccggcagc cccgccatta agaagggcat cctgcagaca
     2281 gtgaaggtgg tggacgagct cgtgaaagtg atgggccggc acaagcccga gaacatcgtg
     2341 atcgaaatgg ccagagagaa ccagaccacc cagaagggac agaagaacag ccgcgagaga
     2401 atgaagcgga tcgaagaggg catcaaagag ctgggcagcc agatcctgaa agaacacccc
     2461 gtggaaaaca cccagctgca gaacgagaag ctgtacctgt actacctgca gaatgggcgg
     2521 gatatgtacg tggaccagga actggacatc aaccggctgt ccgactacga tgtggacgcc
     2581 atcgtgcctc agagctttct gaaggacgac tccatcgaca acaaggtgct gaccagaagc
     2641 gacaagaacc ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa gaagatgaag
     2701 aactactggc ggcagctgct gaacgccaag ctgattaccc agagaaagtt cgacaatctg
     2761 accaaggccg agagaggcgg cctgagcgaa ctggataagg ccggcttcat caagagacag
     2821 ctggtggaaa cccggcagat cacaaagcac gtggcacaga tcctggactc ccggatgaac
     2881 actaagtacg acgagaatga caagctgatc cgggaagtga aagtgatcac cctgaagtcc
     2941 aagctggtgt ccgatttccg gaaggatttc cagttttaca aagtgcgcga gatcaacaac
     3001 taccaccacg cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag
     3061 taccctaagc tggaaagcga gttcgtgtac ggcgactaca aggtgtacga cgtgcggaag
     3121 atgatcgcca agagcgagca ggaaatcggc aaggctaccg ccaagtactt cttctacagc
     3181 aacatcatga actttttcaa gaccgagatt accctggcca acggcgagat ccggaagcgg
     3241 cctctgatcg agacaaacgg cgaaaccggg gagatcgtgt gggataaggg ccgggatttt
     3301 gccaccgtgc ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa gaccgaggtg
     3361 cagacaggcg gcttcagcaa agagtctatc ctgcccaaga ggaacagcga taagctgatc
     3421 gccagaaaga aggactggga ccctaagaag tacggcggct tcgacagccc caccgtggcc
     3481 tattctgtgc tggtggtggc caaagtggaa aagggcaagt ccaagaaact gaagagtgtg
     3541 aaagagctgc tggggatcac catcatggaa agaagcagct tcgagaagaa tcccatcgac
     3601 tttctggaag ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa gctgcctaag
     3661 tactccctgt tcgagctgga aaacggccgg aagagaatgc tggcctctgc cggcgaactg
     3721 cagaagggaa acgaactggc cctgccctcc aaatatgtga acttcctgta cctggccagc
     3781 cactatgaga agctgaaggg ctcccccgag gataatgagc agaaacagct gtttgtggaa
     3841 cagcacaagc actacctgga cgagatcatc gagcagatca gcgagttctc caagagagtg
     3901 atcctggccg acgctaatct ggacaaagtg ctgtccgcct acaacaagca ccgggataag
     3961 cccatcagag agcaggccga gaatatcatc cacctgttta ccctgaccaa tctgggagcc
     4021 cctgccgcct tcaagtactt tgacaccacc atcgaccgga agaggtacac cagcaccaaa
     4081 gaggtgctgg acgccaccct gatccaccag agcatcaccg gcctgtacga gacacggatc
     4141 gacctgtctc agctgggagg cgacaaaagg ccggcggcca cgaaaaaggc cggccaggca
     4201 aaaaagaaaa agtaa
//
