LOCUS       sequence-260232-        4167 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Expression of fusion protein M3-M14-dCas9 in Mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 4167)
  AUTHORS   Liu XM, Zhou J, Mao Y, Ji Q, Qian SB
  TITLE     Programmable RNA N(6)-methyladenosine editing by CRISPR-Cas9 
            conjugates.
  JOURNAL   Nat Chem Biol. 2019 Sep;15(9):865-871. doi: 
            10.1038/s41589-019-0327-1. Epub 2019 Aug 5.
  PUBMED    31383972
REFERENCE   2  (bases 1 to 4167)
  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"; doi: 
            "10.1038/s41589-019-0327-1"; journalName: "Nat Chem Biol"; date: 
            "2019-09"; volume: "15"; issue: "9"; pages: "865-871"
COMMENT     Sequence Label: M3-M14-dCas9
FEATURES             Location/Qualifiers
     source          1..4167
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     CDS             join(67..3465,3466..3468,3469..4065,4066,4067..4069,
                     4070..4071,4072..4074,4075..4167)
                     /codon_start=1
                     /product="Streptococcus pyogenes Cas9 endonuclease with the
                     D1135V/R1335Q/T1337R mutations"
                     /label=Cas9 VQR
                     /note="has an altered recognition specificity for the 
                     protospacer adjacent motif (PAM) (Kleinstiver et al., 
                     2015)"
                     /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(67..90,91..93,94..2580,2581..2583,2584..4167)
                     /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"
ORIGIN
        1 aaatctaaat ctgaccgaag cggcagcgag actcccggga cctcagagtc cgccacaccc
       61 gaaagtgata aaaagtattc tattggttta gccatcggca ctaattccgt tggatgggct
      121 gtcataaccg atgaatacaa agtaccttca aagaaattta aggtgttggg gaacacagac
      181 cgtcattcga ttaaaaagaa tcttatcggt gccctcctat tcgatagtgg cgaaacggca
      241 gaggcgactc gcctgaaacg aaccgctcgg agaaggtata cacgtcgcaa gaaccgaata
      301 tgttacttac aagaaatttt tagcaatgag atggccaaag ttgacgattc tttctttcac
      361 cgtttggaag agtccttcct tgtcgaagag gacaagaaac atgaacggca ccccatcttt
      421 ggaaacatag tagatgaggt ggcatatcat gaaaagtacc caacgattta tcacctcaga
      481 aaaaagctag ttgactcaac tgataaagcg gacctgaggt taatctactt ggctcttgcc
      541 catatgataa agttccgtgg gcactttctc attgagggtg atctaaatcc ggacaactcg
      601 gatgtcgaca aactgttcat ccagttagta caaacctata atcagttgtt tgaagagaac
      661 cctataaatg caagtggcgt ggatgcgaag gctattctta gcgcccgcct ctctaaatcc
      721 cgacggctag aaaacctgat cgcacaatta cccggagaga agaaaaatgg gttgttcggt
      781 aaccttatag cgctctcact aggcctgaca ccaaatttta agtcgaactt cgacttagct
      841 gaagatgcca aattgcagct tagtaaggac acgtacgatg acgatctcga caatctactg
      901 gcacaaattg gagatcagta tgcggactta tttttggctg ccaaaaacct tagcgatgca
      961 atcctcctat ctgacatact gagagttaat actgagatta ccaaggcgcc gttatccgct
     1021 tcaatgatca aaaggtacga tgaacatcac caagacttga cacttctcaa ggccctagtc
     1081 cgtcagcaac tgcctgagaa atataaggaa atattctttg atcagtcgaa aaacgggtac
     1141 gcaggttata ttgacggcgg agcgagtcaa gaggaattct acaagtttat caaacccata
     1201 ttagagaaga tggatgggac ggaagagttg cttgtaaaac tcaatcgcga agatctactg
     1261 cgaaagcagc ggactttcga caacggtagc attccacatc aaatccactt aggcgaattg
     1321 catgctatac ttagaaggca ggaggatttt tatccgttcc tcaaagacaa tcgtgaaaag
     1381 attgagaaaa tcctaacctt tcgcatacct tactatgtgg gacccctggc ccgagggaac
     1441 tctcggttcg catggatgac aagaaagtcc gaagaaacga ttactccatg gaattttgag
     1501 gaagttgtcg ataaaggtgc gtcagctcaa tcgttcatcg agaggatgac caactttgac
     1561 aagaatttac cgaacgaaaa agtattgcct aagcacagtt tactttacga gtatttcaca
     1621 gtgtacaatg aactcacgaa agttaagtat gtcactgagg gcatgcgtaa acccgccttt
     1681 ctaagcggag aacagaagaa agcaatagta gatctgttat tcaagaccaa ccgcaaagtg
     1741 acagttaagc aattgaaaga ggactacttt aagaaaattg aatgcttcga ttctgtcgag
     1801 atctccgggg tagaagatcg atttaatgcg tcacttggta cgtatcatga cctcctaaag
     1861 ataattaaag ataaggactt cctggataac gaagagaatg aagatatctt agaagatata
     1921 gtgttgactc ttaccctctt tgaagatcgg gaaatgattg aggaaagact aaaaacatac
     1981 gctcacctgt tcgacgataa ggttatgaaa cagttaaaga ggcgtcgcta tacgggctgg
     2041 ggacgattgt cgcggaaact tatcaacggg ataagagaca agcaaagtgg taaaactatt
     2101 ctcgattttc taaagagcga cggcttcgcc aataggaact ttatgcagct gatccatgat
     2161 gactctttaa ccttcaaaga ggatatacaa aaggcacagg tttccggaca aggggactca
     2221 ttgcacgaac atattgcgaa tcttgctggt tcgccagcca tcaaaaaggg catactccag
     2281 acagtcaaag tagtggatga gctagttaag gtcatgggac gtcacaaacc ggaaaacatt
     2341 gtaatcgaga tggcacgcga aaatcaaacg actcagaagg ggcaaaaaaa cagtcgagag
     2401 cggatgaaga gaatagaaga gggtattaaa gaactgggca gccagatctt aaaggagcat
     2461 cctgtggaaa atacccaatt gcagaacgag aaactttacc tctattacct acaaaatgga
     2521 agggacatgt atgttgatca ggaactggac ataaaccgtt tatctgatta cgacgtcgat
     2581 gccattgtac cccaatcctt tttgaaggac gattcaatcg acaataaagt gcttacacgc
     2641 tcggataaga accgagggaa aagtgacaat gttccaagcg aggaagtcgt aaagaaaatg
     2701 aagaactatt ggcggcagct cctaaatgcg aaactgataa cgcaaagaaa gttcgataac
     2761 ttaactaaag ctgagagggg tggcttgtct gaacttgaca aggccggatt tattaaacgt
     2821 cagctcgtgg aaacccgcca aatcacaaag catgttgcac agatactaga ttcccgaatg
     2881 aatacgaaat acgacgagaa cgataagctg attcgggaag tcaaagtaat cactttaaag
     2941 tcaaaattgg tgtcggactt cagaaaggat tttcaattct ataaagttag ggagataaat
     3001 aactaccacc atgcgcacga cgcttatctt aatgccgtcg tagggaccgc actcattaag
     3061 aaatacccga agctagaaag tgagtttgtg tatggtgatt acaaagttta tgacgtccgt
     3121 aagatgatcg cgaaaagcga acaggagata ggcaaggcta cagccaaata cttcttttat
     3181 tctaacatta tgaatttctt taagacggaa atcactctgg caaacggaga gatacgcaaa
     3241 cgacctttaa ttgaaaccaa tggggagaca ggtgaaatcg tatgggataa gggccgggac
     3301 ttcgcgacgg tgagaaaagt tttgtccatg ccccaagtca acatagtaaa gaaaactgag
     3361 gtgcagaccg gagggttttc aaaggaatcg attcttccaa aaaggaatag tgataagctc
     3421 atcgctcgta aaaaggactg ggacccgaaa aagtacggtg gcttcgatag ccctacagtt
     3481 gcctattctg tcctagtagt ggcaaaagtt gagaagggaa aatccaagaa actgaagtca
     3541 gtcaaagaat tattggggat aacgattatg gagcgctcgt cttttgaaaa gaaccccatc
     3601 gacttccttg aggcgaaagg ttacaaggaa gtaaaaaagg atctcataat taaactacca
     3661 aagtatagtc tgtttgagtt agaaaatggc cgaaaacgga tgttggctag cgccggagag
     3721 cttcaaaagg ggaacgaact cgcactaccg tctaaatacg tgaatttcct gtatttagcg
     3781 tcccattacg agaagttgaa aggttcacct gaagataacg aacagaagca actttttgtt
     3841 gagcagcaca aacattatct cgacgaaatc atagagcaaa tttcggaatt cagtaagaga
     3901 gtcatcctag ctgatgccaa tctggacaaa gtattaagcg catacaacaa gcacagggat
     3961 aaacccatac gtgagcaggc ggaaaatatt atccatttgt ttactcttac caacctcggc
     4021 gctccagccg cattcaagta ttttgacaca acgatagatc gcaaacgata cacttctacc
     4081 aaggaggtgc tagacgcgac actgattcac caatccatca cgggattata tgaaactcgg
     4141 atagatttgt cacagcttgg gggtgac
//
