LOCUS       sequence-362131-        7599 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of nuclease inactive 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: pAT15541_dCBE-SuperFi-Cas9
FEATURES             Location/Qualifiers
     source          1..7599
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(146..937,938..1006))
                     /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     769..788
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1007..1111)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        1194..1405
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      1425..1434
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1434..1499
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1506..1526
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1551..2237
                     /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             join(2286..2309,2310..2312,2313..4868,4869..4871,
                     4872..6386)
                     /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 N863A mutation in the HNH 
                     catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVDGDDKVDDDDKMIAKSEDEIGDATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             6399..6647
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             6660..6680
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(6708..6725)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6714..6921
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      complement(6986..7574)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7066..7085)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
       61 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
      121 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
      181 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
      241 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
      301 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
      361 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
      421 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
      481 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
      541 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
      601 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
      661 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
      721 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
      781 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
      841 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
      901 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
      961 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     1021 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     1081 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
     1141 tccagcagag atccactttg gcgccggctc gagtggctcc ggtgcccgtc agtgggcaga
     1201 gcgcacatcg cccacagtcc ccgagaagtt ggggggaggg gtcggcaatt gaaccggtgc
     1261 ctagagaagg tggcgcgggg taaactggga aagtgatgtc gtgtactggc tccgcctttt
     1321 tcccgagggt gggggagaac cgtatataag tgcagtagtc gccgtgaacg ttctttttcg
     1381 caacgggttt gccgccagaa cacaggtgtc gtgacgcggg atccgccacc atggactata
     1441 aggaccacga cggagactac aaggatcatg atattgatta caaagacgat gacgataaga
     1501 tggccccaaa gaagaagcgg aaggtcggta tccacggagt cccagcagcc atgagctcag
     1561 agactggccc agtggctgtg gaccccacat tgagacggcg gatcgagccc catgagtttg
     1621 aggtattctt cgatccgaga gagctccgca aggagacctg cctgctttac gaaattaatt
     1681 gggggggccg gcactccatt tggcgacata catcacagaa cactaacaag cacgtcgaag
     1741 tcaacttcat cgagaagttc acgacagaaa gatatttctg tccgaacaca aggtgcagca
     1801 ttacctggtt tctcagctgg agcccatgcg gcgaatgtag tagggccatc actgaattcc
     1861 tgtcaaggta tccccacgtc actctgttta tttacatcgc aaggctgtac caccacgctg
     1921 acccccgcaa tcgacaaggc ctgcgggatt tgatctcttc aggtgtgact atccaaatta
     1981 tgactgagca ggagtcagga tactgctgga gaaactttgt gaattatagc ccgagtaatg
     2041 aagcccactg gcctaggtat ccccatctgt gggtacgact gtacgttctt gaactgtact
     2101 gcatcatact gggcctgcct ccttgtctca acattctgag aaggaagcag ccacagctga
     2161 cattctttac catcgctctt cagtcttgtc attaccagcg actgccccca cacattctct
     2221 gggccaccgg gttgaaaagc ggcagcgaga ctcccgggac ctcagagtcc gccacacccg
     2281 aaagtgacaa gaagtacagc atcggcctgg ccatcggcac caactctgtg ggctgggccg
     2341 tgatcaccga cgagtacaag gtgcccagca agaaattcaa ggtgctgggc aacaccgacc
     2401 ggcacagcat caagaagaac ctgatcggag ccctgctgtt cgacagcggc gaaacagccg
     2461 aggccacccg gctgaagaga accgccagaa gaagatacac cagacggaag aaccggatct
     2521 gctatctgca agagatcttc agcaacgaga tggccaaggt ggacgacagc ttcttccaca
     2581 gactggaaga gtccttcctg gtggaagagg ataagaagca cgagcggcac cccatcttcg
     2641 gcaacatcgt ggacgaggtg gcctaccacg agaagtaccc caccatctac cacctgagaa
     2701 agaaactggt ggacagcacc gacaaggccg acctgcggct gatctatctg gccctggccc
     2761 acatgatcaa gttccggggc cacttcctga tcgagggcga cctgaacccc gacaacagcg
     2821 acgtggacaa gctgttcatc cagctggtgc agacctacaa ccagctgttc gaggaaaacc
     2881 ccatcaacgc cagcggcgtg gacgccaagg ccatcctgtc tgccagactg agcaagagca
     2941 gacggctgga aaatctgatc gcccagctgc ccggcgagaa gaagaatggc ctgttcggca
     3001 acctgattgc cctgagcctg ggcctgaccc ccaacttcaa gagcaacttc gacctggccg
     3061 aggatgccaa actgcagctg agcaaggaca cctacgacga cgacctggac aacctgctgg
     3121 cccagatcgg cgaccagtac gccgacctgt ttctggccgc caagaacctg tccgacgcca
     3181 tcctgctgag cgacatcctg agagtgaaca ccgagatcac caaggccccc ctgagcgcct
     3241 ctatgatcaa gagatacgac gagcaccacc aggacctgac cctgctgaaa gctctcgtgc
     3301 ggcagcagct gcctgagaag tacaaagaga ttttcttcga ccagagcaag aacggctacg
     3361 ccggctacat tgacggcgga gccagccagg aagagttcta caagttcatc aagcccatcc
     3421 tggaaaagat ggacggcacc gaggaactgc tcgtgaagct gaacagagag gacctgctgc
     3481 ggaagcagcg gaccttcgac aacggcagca tcccccacca gatccacctg ggagagctgc
     3541 acgccattct gcggcggcag gaagattttt acccattcct gaaggacaac cgggaaaaga
     3601 tcgagaagat cctgaccttc cgcatcccct actacgtggg ccctctggcc aggggaaaca
     3661 gcagattcgc ctggatgacc agaaagagcg aggaaaccat caccccctgg aacttcgagg
     3721 aagtggtgga caagggcgct tccgcccaga gcttcatcga gcggatgacc aacttcgata
     3781 agaacctgcc caacgagaag gtgctgccca agcacagcct gctgtacgag tacttcaccg
     3841 tgtataacga gctgaccaaa gtgaaatacg tgaccgaggg aatgagaaag cccgccttcc
     3901 tgagcggcga gcagaaaaag gccatcgtgg acctgctgtt caagaccaac cggaaagtga
     3961 ccgtgaagca gctgaaagag gactacttca agaaaatcga gtgcttcgac tccgtggaaa
     4021 tctccggcgt ggaagatcgg ttcaacgcct ccctgggcac ataccacgat ctgctgaaaa
     4081 ttatcaagga caaggacttc ctggacaatg aggaaaacga ggacattctg gaagatatcg
     4141 tgctgaccct gacactgttt gaggacagag agatgatcga ggaacggctg aaaacctatg
     4201 cccacctgtt cgacgacaaa gtgatgaagc agctgaagcg gcggagatac accggctggg
     4261 gcaggctgag ccggaagctg atcaacggca tccgggacaa gcagtccggc aagacaatcc
     4321 tggatttcct gaagtccgac ggcttcgcca acagaaactt catgcagctg atccacgacg
     4381 acagcctgac ctttaaagag gacatccaga aagcccaggt gtccggccag ggcgatagcc
     4441 tgcacgagca cattgccaat ctggccggca gccccgccat taagaagggc atcctgcaga
     4501 cagtgaaggt ggtggacgag ctcgtgaaag tgatgggccg gcacaagccc gagaacatcg
     4561 tgatcgaaat ggccagagag aaccagacca cccagaaggg acagaagaac agccgcgaga
     4621 gaatgaagcg gatcgaagag ggcatcaaag agctgggcag ccagatcctg aaagaacacc
     4681 cagtggaaaa cacccagctg cagaacgaga agctgtacct gtactacctg cagaatgggc
     4741 gggatatgta cgtggaccag gaactggaca tcaaccggct gtccgactac gatgtggacg
     4801 ccatcgtgcc tcagagcttt ctgaaggacg actccatcga caacaaggtg ctgaccagaa
     4861 gcgacaagaa ccggggcaag agcgacaacg tgccctccga agaggtcgtg aagaagatga
     4921 agaactactg gcggcagctg ctgaacgcca agctgattac ccagagaaag ttcgacaatc
     4981 tgaccaaggc cgagagaggc ggcctgagcg aactggataa ggccggcttc atcaagagac
     5041 agctggtgga aacccggcag atcacaaagc acgtggcaca gatcctggac tcccggatga
     5101 acactaagta cgacgagaat gacaagctga tccgggaagt gaaagtgatc accctgaagt
     5161 ccaagctggt gtccgatttc cggaaggatt tccagtttta caaagtgcgc gagatcaaca
     5221 actaccacca cgcccacgac gcgtacctga acgccgtcgt gggaaccgcc ctgatcaaaa
     5281 agtaccctaa gctggaaagc gagttcgtgg acggcgacga caaggtggac gacgacgaca
     5341 agatgatcgc caagagcgaa gacgaaatcg gcgacgctac cgccaagtac ttcttctaca
     5401 gcaacatcat gaactttttc aagaccgaga ttaccctggc caacggcgag atccggaagc
     5461 ggcctctgat cgagacaaac ggcgaaaccg gggagatcgt gtgggataag ggccgggatt
     5521 ttgccaccgt gcggaaagtg ctgagcatgc cccaagtgaa tatcgtgaaa aagaccgagg
     5581 tgcagacagg cggcttcagc aaagagtcta tcctgcccaa gaggaacagc gataagctga
     5641 tcgccagaaa gaaggactgg gaccctaaga agtacggcgg cttcgacagc cccaccgtgg
     5701 cctattctgt gctggtggtg gccaaagtgg aaaagggcaa gtccaagaaa ctgaagagtg
     5761 tgaaagagct gctggggatc accatcatgg aaagaagcag cttcgagaag aatcccatcg
     5821 actttctgga agccaagggc tacaaagaag tgaaaaagga cctgatcatc aagctgccta
     5881 agtactccct gttcgagctg gaaaacggcc ggaagagaat gctggcctct gccggcgaac
     5941 tgcagaaggg aaacgaactg gccctgccct ccaaatatgt gaacttcctg tacctggcca
     6001 gccactatga gaagctgaag ggctcccccg aggataatga gcagaaacag ctgtttgtgg
     6061 aacagcacaa gcactacctg gacgagatca tcgagcagat cagcgagttc tccaagagag
     6121 tgatcctggc cgacgctaat ctggacaaag tgctgtccgc ctacaacaag caccgggata
     6181 agcccatcag agagcaggcc gagaatatca tccacctgtt taccctgacc aatctgggag
     6241 cccctgccgc cttcaagtac tttgacacca ccatcgaccg gaagaggtac accagcacca
     6301 aagaggtgct ggacgccacc ctgatccacc agagcatcac cggcctgtac gagacacgga
     6361 tcgacctgtc tcagctggga ggcgattcag gcggatctac taatctgtca gatattattg
     6421 aaaaggagac cggtaagcaa ctggttatcc aggaatccat cctcatgctc ccagaggagg
     6481 tggaagaagt cattgggaac aagccggaaa gcgatatact cgtgcacacc gcctacgacg
     6541 agagcaccga cgagaatgtc atgcttctga ctagcgacgc ccctgaatac aagccttggg
     6601 ctctggtcat acaggatagc aacggtgaga acaagattaa gatgctctct ggtggttctc
     6661 ccaagaagaa gaggaaagtc tgacatgtcc tagagctcgc tgatcagcct cgactgtgcc
     6721 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     6781 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     6841 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     6901 gaatagcagg catgctgggg agcggccgct gagcaaaagg ccagcaaaag gccaggaacc
     6961 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     7021 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     7081 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     7141 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     7201 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     7261 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     7321 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     7381 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     7441 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     7501 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     7561 aaaaaggatc tcaagaagat cctttgatct tttctacgg
//
