LOCUS       sequence-362132-        7599 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of SuperFi-Cas9 CBE base editor .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7599)
  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 7599)
  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: pAT15542_nCBE-SuperFi-Cas9
FEATURES             Location/Qualifiers
     source          1..7599
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        68..279
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      299..308
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             308..373
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             380..400
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             425..1111
                     /codon_start=1
                     /product="cytidine deaminase (C to U editing enzyme) from 
                     rat"
                     /label=APOBEC-1
                     /note="can use ssDNA as a substrate (Komor et al., 2016)"
                     /translation="MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWG
                     GRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFL
                     SRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSN
                     EAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHI
                     LWATGLK"
     CDS             1160..5260
                     /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="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVDGDDKVDDDDKMIAKSEDEIGDATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             5273..5521
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             5534..5554
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(5582..5599)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5588..5795
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      complement(5860..6448)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5940..5959)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6619..7410,7411..7479))
                     /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     7242..7261
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7480..7584)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 agtgccacct gacgtccagc agagatccac tttggcgccg gctcgagtgg ctccggtgcc
       61 cgtcagtggg cagagcgcac atcgcccaca gtccccgaga agttgggggg aggggtcggc
      121 aattgaaccg gtgcctagag aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac
      181 tggctccgcc tttttcccga gggtggggga gaaccgtata taagtgcagt agtcgccgtg
      241 aacgttcttt ttcgcaacgg gtttgccgcc agaacacagg tgtcgtgacg cgggatccgc
      301 caccatggac tataaggacc acgacggaga ctacaaggat catgatattg attacaaaga
      361 cgatgacgat aagatggccc caaagaagaa gcggaaggtc ggtatccacg gagtcccagc
      421 agccatgagc tcagagactg gcccagtggc tgtggacccc acattgagac ggcggatcga
      481 gccccatgag tttgaggtat tcttcgatcc gagagagctc cgcaaggaga cctgcctgct
      541 ttacgaaatt aattgggggg gccggcactc catttggcga catacatcac agaacactaa
      601 caagcacgtc gaagtcaact tcatcgagaa gttcacgaca gaaagatatt tctgtccgaa
      661 cacaaggtgc agcattacct ggtttctcag ctggagccca tgcggcgaat gtagtagggc
      721 catcactgaa ttcctgtcaa ggtatcccca cgtcactctg tttatttaca tcgcaaggct
      781 gtaccaccac gctgaccccc gcaatcgaca aggcctgcgg gatttgatct cttcaggtgt
      841 gactatccaa attatgactg agcaggagtc aggatactgc tggagaaact ttgtgaatta
      901 tagcccgagt aatgaagccc actggcctag gtatccccat ctgtgggtac gactgtacgt
      961 tcttgaactg tactgcatca tactgggcct gcctccttgt ctcaacattc tgagaaggaa
     1021 gcagccacag ctgacattct ttaccatcgc tcttcagtct tgtcattacc agcgactgcc
     1081 cccacacatt ctctgggcca ccgggttgaa aagcggcagc gagactcccg ggacctcaga
     1141 gtccgccaca cccgaaagtg acaagaagta cagcatcggc ctggccatcg gcaccaactc
     1201 tgtgggctgg gccgtgatca ccgacgagta caaggtgccc agcaagaaat tcaaggtgct
     1261 gggcaacacc gaccggcaca gcatcaagaa gaacctgatc ggagccctgc tgttcgacag
     1321 cggcgaaaca gccgaggcca cccggctgaa gagaaccgcc agaagaagat acaccagacg
     1381 gaagaaccgg atctgctatc tgcaagagat cttcagcaac gagatggcca aggtggacga
     1441 cagcttcttc cacagactgg aagagtcctt cctggtggaa gaggataaga agcacgagcg
     1501 gcaccccatc ttcggcaaca tcgtggacga ggtggcctac cacgagaagt accccaccat
     1561 ctaccacctg agaaagaaac tggtggacag caccgacaag gccgacctgc ggctgatcta
     1621 tctggccctg gcccacatga tcaagttccg gggccacttc ctgatcgagg gcgacctgaa
     1681 ccccgacaac agcgacgtgg acaagctgtt catccagctg gtgcagacct acaaccagct
     1741 gttcgaggaa aaccccatca acgccagcgg cgtggacgcc aaggccatcc tgtctgccag
     1801 actgagcaag agcagacggc tggaaaatct gatcgcccag ctgcccggcg agaagaagaa
     1861 tggcctgttc ggcaacctga ttgccctgag cctgggcctg acccccaact tcaagagcaa
     1921 cttcgacctg gccgaggatg ccaaactgca gctgagcaag gacacctacg acgacgacct
     1981 ggacaacctg ctggcccaga tcggcgacca gtacgccgac ctgtttctgg ccgccaagaa
     2041 cctgtccgac gccatcctgc tgagcgacat cctgagagtg aacaccgaga tcaccaaggc
     2101 ccccctgagc gcctctatga tcaagagata cgacgagcac caccaggacc tgaccctgct
     2161 gaaagctctc gtgcggcagc agctgcctga gaagtacaaa gagattttct tcgaccagag
     2221 caagaacggc tacgccggct acattgacgg cggagccagc caggaagagt tctacaagtt
     2281 catcaagccc atcctggaaa agatggacgg caccgaggaa ctgctcgtga agctgaacag
     2341 agaggacctg ctgcggaagc agcggacctt cgacaacggc agcatccccc accagatcca
     2401 cctgggagag ctgcacgcca ttctgcggcg gcaggaagat ttttacccat tcctgaagga
     2461 caaccgggaa aagatcgaga agatcctgac cttccgcatc ccctactacg tgggccctct
     2521 ggccagggga aacagcagat tcgcctggat gaccagaaag agcgaggaaa ccatcacccc
     2581 ctggaacttc gaggaagtgg tggacaaggg cgcttccgcc cagagcttca tcgagcggat
     2641 gaccaacttc gataagaacc tgcccaacga gaaggtgctg cccaagcaca gcctgctgta
     2701 cgagtacttc accgtgtata acgagctgac caaagtgaaa tacgtgaccg agggaatgag
     2761 aaagcccgcc ttcctgagcg gcgagcagaa aaaggccatc gtggacctgc tgttcaagac
     2821 caaccggaaa gtgaccgtga agcagctgaa agaggactac ttcaagaaaa tcgagtgctt
     2881 cgactccgtg gaaatctccg gcgtggaaga tcggttcaac gcctccctgg gcacatacca
     2941 cgatctgctg aaaattatca aggacaagga cttcctggac aatgaggaaa acgaggacat
     3001 tctggaagat atcgtgctga ccctgacact gtttgaggac agagagatga tcgaggaacg
     3061 gctgaaaacc tatgcccacc tgttcgacga caaagtgatg aagcagctga agcggcggag
     3121 atacaccggc tggggcaggc tgagccggaa gctgatcaac ggcatccggg acaagcagtc
     3181 cggcaagaca atcctggatt tcctgaagtc cgacggcttc gccaacagaa acttcatgca
     3241 gctgatccac gacgacagcc tgacctttaa agaggacatc cagaaagccc aggtgtccgg
     3301 ccagggcgat agcctgcacg agcacattgc caatctggcc ggcagccccg ccattaagaa
     3361 gggcatcctg cagacagtga aggtggtgga cgagctcgtg aaagtgatgg gccggcacaa
     3421 gcccgagaac atcgtgatcg aaatggccag agagaaccag accacccaga agggacagaa
     3481 gaacagccgc gagagaatga agcggatcga agagggcatc aaagagctgg gcagccagat
     3541 cctgaaagaa cacccagtgg aaaacaccca gctgcagaac gagaagctgt acctgtacta
     3601 cctgcagaat gggcgggata tgtacgtgga ccaggaactg gacatcaacc ggctgtccga
     3661 ctacgatgtg gaccatatcg tgcctcagag ctttctgaag gacgactcca tcgacaacaa
     3721 ggtgctgacc agaagcgaca agaaccgggg caagagcgac aacgtgccct ccgaagaggt
     3781 cgtgaagaag atgaagaact actggcggca gctgctgaac gccaagctga ttacccagag
     3841 aaagttcgac aatctgacca aggccgagag aggcggcctg agcgaactgg ataaggccgg
     3901 cttcatcaag agacagctgg tggaaacccg gcagatcaca aagcacgtgg cacagatcct
     3961 ggactcccgg atgaacacta agtacgacga gaatgacaag ctgatccggg aagtgaaagt
     4021 gatcaccctg aagtccaagc tggtgtccga tttccggaag gatttccagt tttacaaagt
     4081 gcgcgagatc aacaactacc accacgccca cgacgcgtac ctgaacgccg tcgtgggaac
     4141 cgccctgatc aaaaagtacc ctaagctgga aagcgagttc gtggacggcg acgacaaggt
     4201 ggacgacgac gacaagatga tcgccaagag cgaagacgaa atcggcgacg ctaccgccaa
     4261 gtacttcttc tacagcaaca tcatgaactt tttcaagacc gagattaccc tggccaacgg
     4321 cgagatccgg aagcggcctc tgatcgagac aaacggcgaa accggggaga tcgtgtggga
     4381 taagggccgg gattttgcca ccgtgcggaa agtgctgagc atgccccaag tgaatatcgt
     4441 gaaaaagacc gaggtgcaga caggcggctt cagcaaagag tctatcctgc ccaagaggaa
     4501 cagcgataag ctgatcgcca gaaagaagga ctgggaccct aagaagtacg gcggcttcga
     4561 cagccccacc gtggcctatt ctgtgctggt ggtggccaaa gtggaaaagg gcaagtccaa
     4621 gaaactgaag agtgtgaaag agctgctggg gatcaccatc atggaaagaa gcagcttcga
     4681 gaagaatccc atcgactttc tggaagccaa gggctacaaa gaagtgaaaa aggacctgat
     4741 catcaagctg cctaagtact ccctgttcga gctggaaaac ggccggaaga gaatgctggc
     4801 ctctgccggc gaactgcaga agggaaacga actggccctg ccctccaaat atgtgaactt
     4861 cctgtacctg gccagccact atgagaagct gaagggctcc cccgaggata atgagcagaa
     4921 acagctgttt gtggaacagc acaagcacta cctggacgag atcatcgagc agatcagcga
     4981 gttctccaag agagtgatcc tggccgacgc taatctggac aaagtgctgt ccgcctacaa
     5041 caagcaccgg gataagccca tcagagagca ggccgagaat atcatccacc tgtttaccct
     5101 gaccaatctg ggagcccctg ccgccttcaa gtactttgac accaccatcg accggaagag
     5161 gtacaccagc accaaagagg tgctggacgc caccctgatc caccagagca tcaccggcct
     5221 gtacgagaca cggatcgacc tgtctcagct gggaggcgat tcaggcggat ctactaatct
     5281 gtcagatatt attgaaaagg agaccggtaa gcaactggtt atccaggaat ccatcctcat
     5341 gctcccagag gaggtggaag aagtcattgg gaacaagccg gaaagcgata tactcgtgca
     5401 caccgcctac gacgagagca ccgacgagaa tgtcatgctt ctgactagcg acgcccctga
     5461 atacaagcct tgggctctgg tcatacagga tagcaacggt gagaacaaga ttaagatgct
     5521 ctctggtggt tctcccaaga agaagaggaa agtctgacat gtcctagagc tcgctgatca
     5581 gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc
     5641 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg
     5701 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagaa cagcaagggg
     5761 gaggattggg aagagaatag caggcatgct ggggagcggc cgctgagcaa aaggccagca
     5821 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     5881 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     5941 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     6001 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     6061 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     6121 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     6181 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     6241 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     6301 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     6361 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     6421 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     6481 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     6541 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     6601 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     6661 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     6721 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     6781 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     6841 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     6901 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     6961 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     7021 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     7081 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     7141 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     7201 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     7261 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     7321 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     7381 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     7441 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     7501 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     7561 aaataaacaa ataggggttc cgcgcacatt tccccgaaa
//
