LOCUS       sequence-261019-        5337 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Expression of wildtype nSpyCas9 encoding truncated human cytosine 
            deaminase, hAIDx, at the C-terminus..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 5337)
  AUTHORS   Nguyen Tran MT, Mohd Khalid MKN, Wang Q, Walker JKR, Lidgerwood GE, 
            Dilworth KL, Lisowski L, Pebay A, Hewitt AW
  TITLE     Engineering domain-inlaid SaCas9 adenine base editors with reduced 
            RNA off-targets and increased on-target DNA editing.
  JOURNAL   Nat Commun. 2020 Sep 25;11(1):4871. doi: 10.1038/s41467-020-18715-y.
  PUBMED    32978399
REFERENCE   2  (bases 1 to 5337)
  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 
            Commun."; date: "2020-09-25"; volume: "11(1):4871. doi"; pages: " 
            10.1038/s41467-020-18715-y"
COMMENT     Sequence Label: SpyCas9-AIDx
FEATURES             Location/Qualifiers
     source          1..5337
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     regulatory      7..16
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(13..39,40..42,43..2529,2530..2532,2533..4116)
                     /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             4117..4137
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4153..4173
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4795..4842
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4843..4866
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             4876..4932
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
ORIGIN
        1 aagcttgcca ccatggataa aaagtatagc attggtctgg cgattggcac caacagcgtg
       61 ggctgggccg tgattaccga tgagtacaaa gttccgtcca agaagttcaa ggtgctgggt
      121 aacaccgacc gccacagcat caagaaaaac ctgattggtg ccctgctgtt tgatagcggt
      181 gaaaccgccg aggctacccg tctgaagcgc accgcccgtc gccgttatac ccgtcgtaaa
      241 aaccgtatct gctacctgca agagattttc agcaacgaaa tggccaaagt ggacgatagc
      301 ttctttcacc gcctggagga gagcttcctg gtggaggaag acaagaaaca cgagcgtcac
      361 ccgatctttg gcaacattgt ggatgaggtg gcttatcacg aaaagtaccc gaccatctat
      421 cacctgcgta agaaactggt ggatagcacc gacaaggccg atctgcgcct gatctacctg
      481 gcgctggctc acatgatcaa gttccgtggt cactttctga tcgagggcga cctgaacccg
      541 gataacagcg atgtggacaa gctgttcatc cagctggtgc agacctataa ccagctgttt
      601 gaggaaaacc cgattaacgc cagcggtgtg gacgccaagg cgatcctgag cgcgcgtctg
      661 agcaaaagcc gccgtctgga gaacctgatc gcgcagctgc cgggtgaaaa gaaaaacggt
      721 ctgtttggca acctgattgc tctgagcctg ggcctgaccc cgaacttcaa aagcaacttt
      781 gacctggccg aggatgcgaa gctgcaactg agcaaggata cctacgacga tgacctggac
      841 aacctgctgg cgcagatcgg cgaccagtat gctgatctgt tcctggccgc gaagaacctg
      901 agcgatgcga tcctgctgag cgacattctg cgtgtgaaca ccgagattac caaggctccg
      961 ctgagcgcca gcatgatcaa acgctacgat gaacaccacc aggacctgac cctgctgaaa
     1021 gctctggtgc gtcagcagct gccggagaag tataaagaaa tcttctttga ccagagcaag
     1081 aacggttacg ccggctatat tgatggcggt gcgagccagg aggagttcta caagtttatc
     1141 aaaccgattc tggagaaaat ggacggcacc gaggaactgc tggtgaagct gaaccgtgaa
     1201 gatctgctgc gtaaacagcg cacctttgac aacggtagca tcccgcacca gattcacctg
     1261 ggtgagctgc acgccatcct gcgccgtcag gaagatttct acccgtttct gaaggacaac
     1321 cgtgagaaga tcgaaaaaat tctgaccttc cgcattccgt actatgtggg tccgctggct
     1381 cgtggcaaca gccgctttgc ctggatgacc cgcaagagcg aggaaaccat caccccgtgg
     1441 aacttcgagg aagtggtgga caaaggcgcc agcgcccaga gcttcattga gcgtatgacc
     1501 aactttgata agaacctgcc gaacgaaaaa gtgctgccga agcacagcct gctgtacgag
     1561 tatttcaccg tgtataacga actgaccaag gtgaaatacg tgaccgaggg tatgcgcaag
     1621 ccggccttcc tgagcggcga acagaagaaa gcgatcgtgg acctgctgtt taaaaccaac
     1681 cgtaaggtga ccgtgaagca gctgaaagag gattacttca agaaaattga atgcttcgac
     1741 agcgtggaga tcagcggtgt ggaagatcgt tttaacgcga gcctgggcac ctatcacgac
     1801 ctgctgaaaa tcattaagga taaagacttc ctggataacg aggaaaacga ggacatcctg
     1861 gaagatattg tgctgaccct gaccctgttt gaggaccgtg aaatgatcga ggaacgcctg
     1921 aagacctacg cccacctgtt cgatgacaaa gtgatgaaac agctgaagcg ccgtcgctat
     1981 accggttggg gccgtctgag ccgcaaactg atcaacggta ttcgcgataa acagagcggc
     2041 aagaccatcc tggattttct gaagagcgac ggtttcgcga accgtaactt tatgcagctg
     2101 attcacgatg acagcctgac cttcaaagag gacatccaga aggcgcaggt gagcggtcag
     2161 ggcgatagcc tgcacgaaca catcgctaac ctggccggta gcccggccat caagaagggc
     2221 atcctgcaaa ccgtgaaggt ggtggacgag ctggtgaaag tgatgggtcg tcacaagccg
     2281 gaaaacatcg tgattgagat ggcccgcgaa aaccagacca cccagaaagg ccagaagaac
     2341 agccgtgagc gcatgaagcg tatcgaggaa ggcatcaagg aactgggcag ccagatcctg
     2401 aaggagcacc cggtggaaaa cacccagctg caaaacgaga aactgtatct gtactacctg
     2461 caaaacggtc gtgacatgta cgtggatcag gagctggaca ttaaccgcct gagcgattac
     2521 gacgtggatg cgatcgtgcc gcagagcttc ctgaaggatg acagcattga caacaaagtg
     2581 ctgacccgta gcgataaaaa ccgcggcaag agcgacaacg tgccgagcga ggaagtggtg
     2641 aagaaaatga aaaactactg gcgtcagctg ctgaacgcca aactgatcac ccagcgtaag
     2701 tttgacaacc tgaccaaagc tgagcgcggc ggtctgagcg aactggataa agccggtttc
     2761 attaagcgtc agctggtgga aacccgccag atcaccaagc acgtggcgca gattctggac
     2821 agccgcatga acaccaagta cgacgagaac gataaactga tccgtgaagt gaaagtgatc
     2881 accctgaaga gcaaactggt gagcgatttt cgtaaagact tccagtttta caaggtgcgc
     2941 gagatcaaca actatcacca cgcgcacgac gcttacctga acgccgtggt gggcaccgcc
     3001 ctgatcaaga agtatccgaa gctggagagc gagttcgtgt acggcgatta taaagtgtac
     3061 gacgtgcgta aaatgatcgc gaagagcgag caggaaattg gcaaagcgac cgctaagtat
     3121 ttcttttaca gcaacatcat gaacttcttt aagaccgaga tcaccctggc caacggtgaa
     3181 atccgtaaac gcccgctgat tgaaaccaac ggtgaaaccg gcgaaatcgt gtgggataag
     3241 ggccgtgact ttgcgaccgt gcgcaaagtg ctgtccatgc cgcaggtgaa cattgtgaag
     3301 aaaaccgagg tgcagaccgg cggtttcagc aaggaaagca ttctgccgaa acgtaacagc
     3361 gacaagctga tcgcccgcaa gaaagactgg gacccgaaga agtatggcgg tttcgatagc
     3421 ccaaccgtgg cctacagcgt gctggtggtg gctaaagtgg agaagggtaa aagcaagaaa
     3481 ctgaaaagcg tgaaggaact gctgggcatc accattatgg agcgtagcag cttcgaaaag
     3541 aacccgatcg actttctgga ggcgaaaggt tacaaggaag tgaagaaaga tctgatcatc
     3601 aagctgccga agtatagcct gtttgagctg gaaaatggtc gtaaacgtat gctggccagc
     3661 gccggcgagc tgcaaaaggg caacgaactg gcgctgccga gcaaatacgt gaacttcctg
     3721 tatctggcca gccactacga gaagctgaaa ggtagcccgg aggacaacga acagaagcag
     3781 ctgtttgtgg agcagcacaa acactacctg gatgagatca ttgaacagat tagcgagttc
     3841 agcaagcgtg tgatcctggc tgatgccaac ctggacaaag tgctgagcgc ctataacaaa
     3901 caccgtgaca agccgatccg cgagcaggcg gaaaacatca ttcacctgtt caccctgacc
     3961 aacctgggtg cgccggctgc cttcaagtat tttgatacca ccattgaccg taaacgctac
     4021 accagcacca aggaggtgct ggatgctacc ctgatccacc agagcattac cggcctgtac
     4081 gaaacccgta tcgatctgag ccagctgggc ggtgacccga agaaaaagcg taaagtgggt
     4141 agcagcggca gcccgaaaaa gaaacgcaaa gtgggtagcg acgctctgga tgacttcgac
     4201 ctggatatgc tgggcagcga cgccctggat gactttggcg gtggcagcat ggatagcctg
     4261 ctgatgaacc gtcgcaaatt cctgtaccag tttaagaacg tgcgttgggc taaaggccgt
     4321 cgcgaaacct atctgtgcta cgtggtgaaa cgtcgcgata gcgccaccag cttcagcctg
     4381 gactttggtt atctgcgcaa caagaacggc tgccacgtgg agctgctgtt cctgcgttat
     4441 attagcgact gggacctgga cccaggccgt tgctaccgtg tgacctggtt taccagctgg
     4501 agcccgtgct atgattgcgc ccgtcatgtg gctgacttcc tgcgtggtaa cccaaacctg
     4561 agcctgcgta tcttcaccgc gcgcctgtac ttttgcgagg accgtaaggc tgaaccagaa
     4621 ggtctgcgtc gcctgcaccg tgctggcgtg cagatcgcca tcatgacctt caaagattac
     4681 ttttattgct ggaacacctt cgtggagaac cacgaacgca cctttaaggc ctgggagggt
     4741 ctgcacgaaa acagcgtgcg tctgagccgc cagctgcgcc gcatcctgct gccaaagcgt
     4801 ccggcggcta ccaagaaagc cggtcaggcg aagaaaaaga aagactacaa agatgacgat
     4861 gacaagggta gcggcgctac caacttcagc ctgctgaagc aggcgggcga cgtggaggaa
     4921 aatccgggcc cgatggctaa accgctgagc caggaggaaa gcaccctgat cgagcgcgcg
     4981 accgctacca ttaacagcat cccgattagc gaagattata gcgtggccag cgccgcgctg
     5041 agcagcgatg gtcgtatctt caccggcgtg aacgtgtatc actttaccgg tggcccgtgc
     5101 gccgagctgg tggtgctggg caccgccgcc gccgccgccg ccggcaacct gacctgcatc
     5161 gtggcgattg gtaacgaaaa tcgtggcatc ctgagcccgt gcggccgttg ccgccaggtg
     5221 ctgctggacc tgcatccggg catcaaggcg attgtgaagg atagcgacgg ccaaccgacc
     5281 gctgtgggta ttcgtgaact gctgccatcc ggctatgttt gggaaggcta aggtacc
//
