LOCUS       sequence-177906-        4272 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Expression plasmid for human codon-optimized dead/inactive increased
            fidelity HeFSpCas9 (without U6-sgRNA coding sequence).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 4272)
  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 4272)
  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-Flag-dHeFSpCas9
FEATURES             Location/Qualifiers
     source          1..4272
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     CDS             4..69
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             76..96
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(121..2658,2659..2661,2662..3123,3124..3126,3127..3294,
                     3295..3297,3298..4221)
                     /codon_start=1
                     /product="Cas9 endonuclease from the Streptococcus pyogenes
                     Type II CRISPR/Cas system, mutated to improve targeting 
                     specificity (Slaymaker et al., 2016)"
                     /label=eSpCas9(1.1)
                     /note="carries the mutations K848A, K1003A, and R1060A"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTAFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGALSRKLINGIRDKQSGKTILDFLKSDGFANRNFMA
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLADDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRAITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPALESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKAPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             4222..4269
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
ORIGIN
        1 atggactata aggaccacga cggagactac aaggatcatg atattgatta caaagacgat
       61 gacgataaga tggccccaaa gaagaagcgg aaggtcggta tccacggagt cccagcagcc
      121 gacaagaagt acagcatcgg gctagccatc ggcaccaact ctgtgggctg ggccgtgatc
      181 accgacgagt acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac
      241 agcatcaaga agaacctgat cggagccctg ctgttcgaca gcggcgaaac agccgaggcc
      301 acccggctga agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat
      361 ctgcaagaga tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg
      421 gaagagtcct tcctggtgga agaggataag aagcacgagc ggcaccccat cttcggcaac
      481 atcgtggacg aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa
      541 ctggtggaca gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg
      601 atcaagttcc ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg
      661 gacaagctgt tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc
      721 aacgccagcg gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagacgg
      781 ctggaaaatc tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggaaacctg
      841 attgccctga gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat
      901 gccaaactgc agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag
      961 atcggcgacc agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg
     1021 ctgagcgaca tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg
     1081 atcaagagat acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag
     1141 cagctgcctg agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc
     1201 tacattgacg gcggagccag ccaggaagag ttctacaagt tcatcaagcc catcctggaa
     1261 aagatggacg gcaccgagga actgctcgtg aagctgaaca gagaggacct gctgcggaag
     1321 cagcggacct tcgacaacgg cagcatcccc caccagatcc acctgggaga gctgcacgcc
     1381 attctgcggc ggcaggaaga tttttaccca ttcctgaagg acaaccggga aaagatcgag
     1441 aagatcctga ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga
     1501 ttcgcctgga tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg
     1561 gtggacaagg gcgcttccgc ccagagcttc atcgagcgga tgaccgcctt cgataagaac
     1621 ctgcccaacg agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtat
     1681 aacgagctga ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc cttcctgagc
     1741 ggcgagcaga aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg
     1801 aagcagctga aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc
     1861 ggcgtggaag atcggttcaa cgcctccctg ggcacatacc acgatctgct gaaaattatc
     1921 aaggacaagg acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg
     1981 accctgacac tgtttgagga cagagagatg atcgaggaac ggctgaaaac ctatgcccac
     2041 ctgttcgacg acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcgcg
     2101 ctgagccgga agctgatcaa cggcatccgc gacaagcagt ccggcaagac aatcctggat
     2161 ttcctgaagt ccgacggctt cgccaacaga aacttcatgg cgctgatcca cgacgacagc
     2221 ctgaccttta aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcac
     2281 gagcacattg ccaatctcgc cggcagcccc gccattaaga agggcatcct ccagacagtg
     2341 aaggtggtgg acgagctggt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc
     2401 gaaatggcca gagagaacca gaccacccag aagggacaga agaacagtcg cgagagaatg
     2461 aagcggatcg aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg
     2521 gaaaacaccc agctccagaa cgagaagctg tacctgtact acctccagaa tgggcgggat
     2581 atgtacgtgg accaggaact ggacatcaac cggctgtccg actacgatgt ggacgccatc
     2641 gtgcctcaga gctttctggc ggacgactcc atcgacaaca aggtgctgac cagaagcgac
     2701 aagaaccggg gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac
     2761 tactggcggc agctgctgaa cgccaagctg attacccaga gaaagttcga caatctgacc
     2821 aaggccgaga gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg
     2881 gtggaaaccc gggcgatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact
     2941 aagtacgacg agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag
     3001 ctggtgtccg atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac
     3061 caccacgccc acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac
     3121 cctgcgctgg aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg
     3181 atcgccaaga gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac
     3241 atcatgaact ttttcaagac cgagattacc ctggccaacg gcgagatccg gaaggcgcct
     3301 ctgatcgaga caaacggcga aaccggggag atcgtgtggg ataagggccg ggattttgcc
     3361 accgtgcgga aagtgctgag catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag
     3421 acaggcggct tcagcaaaga gtctatcctg cccaagagga acagcgataa gctgatcgcc
     3481 agaaagaagg actgggatcc taagaagtac ggcggcttcg acagccccac cgtggcctat
     3541 tctgtgctgg tggtggccaa agtggaaaag ggcaagtcca agaagcttaa gagtgtgaaa
     3601 gagctgctgg ggatcaccat catggaaaga agcagcttcg agaagaatcc catcgacttt
     3661 ctggaagcca agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac
     3721 tcgctcttcg agctggaaaa cggccggaag agaatgctgg cctctgccgg cgaattgcag
     3781 aagggaaacg aactggctct tccctccaaa tatgtgaact tcctgtacct ggccagccac
     3841 tatgagaagc tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag
     3901 cacaagcact acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc
     3961 ctggccgacg ctaatctgga caaagtgctg tccgcctaca acaagcaccg ggataagccc
     4021 atcagagagc aggccgagaa tatcatccac ctgtttaccc tgaccaatct gggagcccct
     4081 gccgccttca agtactttga caccaccatc gataggaaga ggtacacgtc gaccaaagag
     4141 gtgctggacg ccaccctgat ccaccagagc atcaccggcc tgtacgaaac gcgtatcgac
     4201 ctgtctcagc tgggaggcga caaaaggccg gcggccacga aaaaggccgg ccaggcaaaa
     4261 aagaaaaagt aa
//
