LOCUS       sequence-362129-        8064 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of SuperFi-Cas9 nuclease.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8064)
  AUTHORS   Kulcsar PI, Talas A, Ligeti Z, Krausz SL, Welker E
  TITLE     SuperFi-Cas9 exhibits remarkable fidelity but severely reduced 
            activity yet works effectively with ABE8e.
  JOURNAL   Nat Commun. 2022 Nov 11;13(1):6858. doi: 10.1038/s41467-022-34527-8.
  PUBMED    36369279
REFERENCE   2  (bases 1 to 8064)
  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: "2022-11-11"; volume: "13(1):6858. doi"; pages: " 
            10.1038/s41467-022-34527-8"
COMMENT     Sequence Label: pPIK16045_pX330_Flag-SuperFi-Cas9
FEATURES             Location/Qualifiers
     source          1..8064
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          146..373
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      385..394
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             394..459
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             466..486
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             511..4611
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVDGDDKVDDDDKMIAKSEDEIGDATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             4612..4659
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(4687..4704)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4693..4900
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4909..5049
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4909..5038
                     /label=AAV2 ITR
     rep_origin      5124..5579
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5211..5230)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5421..5442
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5596..5615)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5715..5737
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5775..5793)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5861..5965
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5966..6034,6035..6826)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(6184..6203)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6997..7585
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7486..7505
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        7657..7942
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
ORIGIN
        1 cggggcgggg cggggcgagg ggcggggcgg ggcgaggcgg agaggtgcgg cggcagccaa
       61 tcagagcggc gcgctccgaa agtttccttt tatggcgagg cggcggcggc ggcggcccta
      121 taaaaagcga agcgcgcggc gggcgggagt cgctgcgcgc tgccttcgcc ccgtgccccg
      181 ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg accgcgttac tcccacaggt
      241 gagcgggcgg gacggccctt ctcctccggg ctgtaattag ctgagcaaga ggtaagggtt
      301 taagggatgg ttggttggtg gggtattaat gtttaattac ctggagcacc tgcctgaaat
      361 cacttttttt caggttggac cggtgccacc atggactata aggaccacga cggagactac
      421 aaggatcatg atattgatta caaagacgat gacgataaga tggccccaaa gaagaagcgg
      481 aaggtcggta tccacggagt cccagcagcc gacaagaagt acagcatcgg cctggacatc
      541 ggcaccaact ctgtgggctg ggccgtgatc accgacgagt acaaggtgcc cagcaagaaa
      601 ttcaaggtgc tgggcaacac cgaccggcac agcatcaaga agaacctgat cggagccctg
      661 ctgttcgaca gcggcgaaac agccgaggcc acccggctga agagaaccgc cagaagaaga
      721 tacaccagac ggaagaaccg gatctgctat ctgcaagaga tcttcagcaa cgagatggcc
      781 aaggtggacg acagcttctt ccacagactg gaagagtcct tcctggtgga agaggataag
      841 aagcacgagc ggcaccccat cttcggcaac atcgtggacg aggtggccta ccacgagaag
      901 taccccacca tctaccacct gagaaagaaa ctggtggaca gcaccgacaa ggccgacctg
      961 cggctgatct atctggccct ggcccacatg atcaagttcc ggggccactt cctgatcgag
     1021 ggcgacctga accccgacaa cagcgacgtg gacaagctgt tcatccagct ggtgcagacc
     1081 tacaaccagc tgttcgagga aaaccccatc aacgccagcg gcgtggacgc caaggccatc
     1141 ctgtctgcca gactgagcaa gagcagacgg ctggaaaatc tgatcgccca gctgcccggc
     1201 gagaagaaga atggcctgtt cggaaacctg attgccctga gcctgggcct gacccccaac
     1261 ttcaagagca acttcgacct ggccgaggat gccaaactgc agctgagcaa ggacacctac
     1321 gacgacgacc tggacaacct gctggcccag atcggcgacc agtacgccga cctgtttctg
     1381 gccgccaaga acctgtccga cgccatcctg ctgagcgaca tcctgagagt gaacaccgag
     1441 atcaccaagg cccccctgag cgcctctatg atcaagagat acgacgagca ccaccaggac
     1501 ctgaccctgc tgaaagctct cgtgcggcag cagctgcctg agaagtacaa agagattttc
     1561 ttcgaccaga gcaagaacgg ctacgccggc tacattgacg gcggagccag ccaggaagag
     1621 ttctacaagt tcatcaagcc catcctggaa aagatggacg gcaccgagga actgctcgtg
     1681 aagctgaaca gagaggacct gctgcggaag cagcggacct tcgacaacgg cagcatcccc
     1741 caccagatcc acctgggaga gctgcacgcc attctgcggc ggcaggaaga tttttaccca
     1801 ttcctgaagg acaaccggga aaagatcgag aagatcctga ccttccgcat cccctactac
     1861 gtgggccctc tggccagggg aaacagcaga ttcgcctgga tgaccagaaa gagcgaggaa
     1921 accatcaccc cctggaactt cgaggaagtg gtggacaagg gcgcttccgc ccagagcttc
     1981 atcgagcgga tgaccaactt cgataagaac ctgcccaacg agaaggtgct gcccaagcac
     2041 agcctgctgt acgagtactt caccgtgtat aacgagctga ccaaagtgaa atacgtgacc
     2101 gagggaatga gaaagcccgc cttcctgagc ggcgagcaga aaaaggccat cgtggacctg
     2161 ctgttcaaga ccaaccggaa agtgaccgtg aagcagctga aagaggacta cttcaagaaa
     2221 atcgagtgct tcgactccgt ggaaatctcc ggcgtggaag atcggttcaa cgcctccctg
     2281 ggcacatacc acgatctgct gaaaattatc aaggacaagg acttcctgga caatgaggaa
     2341 aacgaggaca ttctggaaga tatcgtgctg accctgacac tgtttgagga cagagagatg
     2401 atcgaggaac ggctgaaaac ctatgcccac ctgttcgacg acaaagtgat gaagcagctg
     2461 aagcggcgga gatacaccgg ctggggcagg ctgagccgga agctgatcaa cggcatccgg
     2521 gacaagcagt ccggcaagac aatcctggat ttcctgaagt ccgacggctt cgccaacaga
     2581 aacttcatgc agctgatcca cgacgacagc ctgaccttta aagaggacat ccagaaagcc
     2641 caggtgtccg gccagggcga tagcctgcac gagcacattg ccaatctggc cggcagcccc
     2701 gccattaaga agggcatcct gcagacagtg aaggtggtgg acgagctcgt gaaagtgatg
     2761 ggccggcaca agcccgagaa catcgtgatc gaaatggcca gagagaacca gaccacccag
     2821 aagggacaga agaacagccg cgagagaatg aagcggatcg aagagggcat caaagagctg
     2881 ggcagccaga tcctgaaaga acaccccgtg gaaaacaccc agctgcagaa cgagaagctg
     2941 tacctgtact acctgcagaa tgggcgggat atgtacgtgg accaggaact ggacatcaac
     3001 cggctgtccg actacgatgt ggaccatatc gtgcctcaga gctttctgaa ggacgactcc
     3061 atcgacaaca aggtgctgac cagaagcgac aagaaccggg gcaagagcga caacgtgccc
     3121 tccgaagagg tcgtgaagaa gatgaagaac tactggcggc agctgctgaa cgccaagctg
     3181 attacccaga gaaagttcga caatctgacc aaggccgaga gaggcggcct gagcgaactg
     3241 gataaggccg gcttcatcaa gagacagctg gtggaaaccc ggcagatcac aaagcacgtg
     3301 gcacagatcc tggactcccg gatgaacact aagtacgacg agaatgacaa gctgatccgg
     3361 gaagtgaaag tgatcaccct gaagtccaag ctggtgtccg atttccggaa ggatttccag
     3421 ttttacaaag tgcgcgagat caacaactac caccacgccc acgacgcgta cctgaacgcc
     3481 gtcgtgggaa ccgccctgat caaaaagtac cctaagctgg aaagcgagtt cgtggacggc
     3541 gacgacaagg tggacgacga cgacaagatg atcgccaaga gcgaagacga aatcggcgac
     3601 gctaccgcca agtacttctt ctacagcaac atcatgaact ttttcaagac cgagattacc
     3661 ctggccaacg gcgagatccg gaagcggcct ctgatcgaga caaacggcga aaccggggag
     3721 atcgtgtggg ataagggccg ggattttgcc accgtgcgga aagtgctgag catgccccaa
     3781 gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct tcagcaaaga gtctatcctg
     3841 cccaagagga acagcgataa gctgatcgcc agaaagaagg actgggaccc taagaagtac
     3901 ggcggcttcg acagccccac cgtggcctat tctgtgctgg tggtggccaa agtggaaaag
     3961 ggcaagtcca agaaactgaa gagtgtgaaa gagctgctgg ggatcaccat catggaaaga
     4021 agcagcttcg agaagaatcc catcgacttt ctggaagcca agggctacaa agaagtgaaa
     4081 aaggacctga tcatcaagct gcctaagtac tccctgttcg agctggaaaa cggccggaag
     4141 agaatgctgg cctctgccgg cgaactgcag aagggaaacg aactggccct gccctccaaa
     4201 tatgtgaact tcctgtacct ggccagccac tatgagaagc tgaagggctc ccccgaggat
     4261 aatgagcaga aacagctgtt tgtggaacag cacaagcact acctggacga gatcatcgag
     4321 cagatcagcg agttctccaa gagagtgatc ctggccgacg ctaatctgga caaagtgctg
     4381 tccgcctaca acaagcaccg ggataagccc atcagagagc aggccgagaa tatcatccac
     4441 ctgtttaccc tgaccaatct gggagcccct gccgccttca agtactttga caccaccatc
     4501 gaccggaaga ggtacaccag caccaaagag gtgctggacg ccaccctgat ccaccagagc
     4561 atcaccggcc tgtacgagac acggatcgac ctgtctcagc tgggaggcga caaaaggccg
     4621 gcggccacga aaaaggccgg ccaggcaaaa aagaaaaagt aagaattcct agagctcgct
     4681 gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc
     4741 cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg
     4801 catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca
     4861 agggggagga ttgggaagag aatagcaggc atgctgggga gcggccgcag gaacccctag
     4921 tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa
     4981 aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcagct
     5041 gcctgcaggg gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     5101 gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     5161 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ttagcgcccg ctcctttcgc
     5221 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     5281 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgattt
     5341 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     5401 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaactctat
     5461 ctcgggctat tcttttgatt tataagggat tttgccgatt tcggtctatt ggttaaaaaa
     5521 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt
     5581 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc
     5641 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
     5701 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
     5761 cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat
     5821 ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     5881 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     5941 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     6001 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     6061 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     6121 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     6181 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
     6241 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     6301 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     6361 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     6421 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     6481 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt
     6541 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     6601 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     6661 atctggagcc ggtgagcgtg gaagccgcgg tatcattgca gcactggggc cagatggtaa
     6721 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     6781 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     6841 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     6901 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     6961 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     7021 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     7081 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     7141 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     7201 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     7261 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     7321 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     7381 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     7441 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     7501 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     7561 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     7621 cttttgctgg ccttttgctc acatgctaga ggtacccgtt acataactta cggtaaatgg
     7681 cccgcctggc tgaccgccca acgacccccg cccattgacg tcaatagtaa cgccaatagg
     7741 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca
     7801 tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc
     7861 ctggcattgt gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt
     7921 attagtcatc gctattacca tggtcgaggt gagccccacg ttctgcttca ctctccccat
     7981 ctcccccccc tccccacccc caattttgta tttatttatt ttttaattat tttgtgcagc
     8041 gatgggggcg gggggggggg gggg
//
