LOCUS       sequence-226909-        8466 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  C-to-T base editor.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8466)
  AUTHORS   Huang TP, Zhao KT, Miller SM, Gaudelli NM, Oakes BL, Fellmann C, 
            Savage DF, Liu DR
  TITLE     Circularly permuted and PAM-modified Cas9 variants broaden the 
            targeting scope of base editors.
  JOURNAL   Nat Biotechnol. 2019 May 20. pii: 10.1038/s41587-019-0134-y. doi: 
            10.1038/s41587-019-0134-y.
  PUBMED    31110355
REFERENCE   2  (bases 1 to 8466)
  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 
            Biotechnol. 2019 May 20. pii: 10.1038/s41587-019-0134-y. doi: 
            10.1038/s41587-019-0134-y."
COMMENT     Sequence Label: CP1028-CBEmax
FEATURES             Location/Qualifiers
     source          1..8466
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        132..335
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     286..306
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     377..396
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        377..395
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             445..465
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             466..1149
                     /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="SSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGG
                     RHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLS
                     RYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNE
                     AHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHIL
                     WATGLK"
     CDS             2329..4047
                     /codon_start=1
                     /product="N-terminal portion of Streptococcus pyogenes Cas9
                     (Zetsche et al., 2015)"
                     /label=Cas9(N)
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIE"
     CDS             5452..5472
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5481..5498
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5521..5538)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5527..5751
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5822..5838)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5822..5838)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5835..5857)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5846..5862
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(5870..5876,5877..5894,5895..5900))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5915..5936
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6053..6070)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6224..6812)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6304..6323)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6983..7774,7775..7843))
                     /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     7606..7625
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7844..7948)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8027..8046)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(8218..8466,1..131)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg
       61 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg
      121 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact
      181 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa
      241 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta
      301 ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct
      361 agagatccgc ggccgctaat acgactcact atagggagag ccgccaccat gaaacggaca
      421 gccgacggaa gcgagttcga gtcaccaaag aagaagcgga aagtctcctc agagactggg
      481 cctgtcgccg tcgatccaac cctgcgccgc cggattgaac ctcacgagtt tgaagtgttc
      541 tttgaccccc gggagctgag aaaggagaca tgcctgctgt acgagatcaa ctggggaggc
      601 aggcactcca tctggaggca cacctctcag aacacaaata agcacgtgga ggtgaacttc
      661 atcgagaagt ttaccacaga gcggtacttc tgccccaata ccagatgtag catcacatgg
      721 tttctgagct ggtccccttg cggagagtgt agcagggcca tcaccgagtt cctgtccaga
      781 tatccacacg tgacactgtt tatctacatc gccaggctgt atcaccacgc agacccaagg
      841 aataggcagg gcctgcgcga tctgatcagc tccggcgtga ccatccagat catgacagag
      901 caggagtccg gctactgctg gcggaacttc gtgaattatt ctcctagcaa cgaggcccac
      961 tggcctaggt acccacacct gtgggtgcgc ctgtacgtgc tggagctgta ttgcatcatc
     1021 ctgggcctgc ccccttgtct gaatatcctg cggagaaagc agccccagct gaccttcttt
     1081 acaatcgccc tgcagtcttg tcactatcag aggctgccac cccacatcct gtgggccaca
     1141 ggcctgaagt ctggaggatc tagcggagga tcctctggca gcgagacacc aggaacaagc
     1201 gagtcagcaa caccagagag cagtggcggc agcagcggcg gcagcgaaat cggcaaggct
     1261 accgccaagt acttcttcta cagcaacatc atgaactttt tcaagaccga gattaccctg
     1321 gccaacggcg agatccggaa gcggcctctg atcgagacaa acggcgaaac cggggagatc
     1381 gtgtgggata agggccggga ttttgccacc gtgcggaaag tgctgagcat gccccaagtg
     1441 aatatcgtga aaaagaccga ggtgcagaca ggcggcttca gcaaagagtc tatcctgccc
     1501 aagaggaaca gcgataagct gatcgccaga aagaaggact gggaccctaa gaagtacggc
     1561 ggcttcgaca gccccaccgt ggcctattct gtgctggtgg tggccaaagt ggaaaagggc
     1621 aagtccaaga aactgaagag tgtgaaagag ctgctgggga tcaccatcat ggaaagaagc
     1681 agcttcgaga agaatcccat cgactttctg gaagccaagg gctacaaaga agtgaaaaag
     1741 gacctgatca tcaagctgcc taagtactcc ctgttcgagc tggaaaacgg ccggaagaga
     1801 atgctggcct ctgccggcga actgcagaag ggaaacgaac tggccctgcc ctccaaatat
     1861 gtgaacttcc tgtacctggc cagccactat gagaagctga agggctcccc cgaggataat
     1921 gagcagaaac agctgtttgt ggaacagcac aagcactacc tggacgagat catcgagcag
     1981 atcagcgagt tctccaagag agtgatcctg gccgacgcta atctggacaa agtgctgtcc
     2041 gcctacaaca agcaccggga taagcccatc agagagcagg ccgagaatat catccacctg
     2101 tttaccctga ccaatctggg agcccctgcc gccttcaagt actttgacac caccatcgac
     2161 cggaagaggt acaccagcac caaagaggtg ctggacgcca ccctgatcca ccagagcatc
     2221 accggcctgt acgagacacg gatcgacctg tctcagctgg gaggcgacgg cggatcagga
     2281 ggctctggcg gttcaggtgg atcaggcggt agcggaggtt caggtggtat ggacaagaag
     2341 tacagcatcg gcctggccat cggcaccaac tctgtgggct gggccgtgat caccgacgag
     2401 tacaaggtgc ccagcaagaa attcaaggtg ctgggcaaca ccgaccggca cagcatcaag
     2461 aagaacctga tcggagccct gctgttcgac agcggcgaaa cagccgaggc cacccggctg
     2521 aagagaaccg ccagaagaag atacaccaga cggaagaacc ggatctgcta tctgcaagag
     2581 atcttcagca acgagatggc caaggtggac gacagcttct tccacagact ggaagagtcc
     2641 ttcctggtgg aagaggataa gaagcacgag cggcacccca tcttcggcaa catcgtggac
     2701 gaggtggcct accacgagaa gtaccccacc atctaccacc tgagaaagaa actggtggac
     2761 agcaccgaca aggccgacct gcggctgatc tatctggccc tggcccacat gatcaagttc
     2821 cggggccact tcctgatcga gggcgacctg aaccccgaca acagcgacgt ggacaagctg
     2881 ttcatccagc tggtgcagac ctacaaccag ctgttcgagg aaaaccccat caacgccagc
     2941 ggcgtggacg ccaaggccat cctgtctgcc agactgagca agagcagacg gctggaaaat
     3001 ctgatcgccc agctgcccgg cgagaagaag aatggcctgt tcggaaacct gattgccctg
     3061 agcctgggcc tgacccccaa cttcaagagc aacttcgacc tggccgagga tgccaaactg
     3121 cagctgagca aggacaccta cgacgacgac ctggacaacc tgctggccca gatcggcgac
     3181 cagtacgccg acctgtttct ggccgccaag aacctgtccg acgccatcct gctgagcgac
     3241 atcctgagag tgaacaccga gatcaccaag gcccccctga gcgcctctat gatcaagaga
     3301 tacgacgagc accaccagga cctgaccctg ctgaaagctc tcgtgcggca gcagctgcct
     3361 gagaagtaca aagagatttt cttcgaccag agcaagaacg gctacgccgg ctacattgac
     3421 ggcggagcca gccaggaaga gttctacaag ttcatcaagc ccatcctgga aaagatggac
     3481 ggcaccgagg aactgctcgt gaagctgaac agagaggacc tgctgcggaa gcagcggacc
     3541 ttcgacaacg gcagcatccc ccaccagatc cacctgggag agctgcacgc cattctgcgg
     3601 cggcaggaag atttttaccc attcctgaag gacaaccggg aaaagatcga gaagatcctg
     3661 accttccgca tcccctacta cgtgggccct ctggccaggg gaaacagcag attcgcctgg
     3721 atgaccagaa agagcgagga aaccatcacc ccctggaact tcgaggaagt ggtggacaag
     3781 ggcgcttccg cccagagctt catcgagcgg atgaccaact tcgataagaa cctgcccaac
     3841 gagaaggtgc tgcccaagca cagcctgctg tacgagtact tcaccgtgta taacgagctg
     3901 accaaagtga aatacgtgac cgagggaatg agaaagcccg ccttcctgag cggcgagcag
     3961 aaaaaggcca tcgtggacct gctgttcaag accaaccgga aagtgaccgt gaagcagctg
     4021 aaagaggact acttcaagaa aatcgagtgc ttcgactccg tggaaatctc cggcgtggaa
     4081 gatcggttca acgcctccct gggcacatac cacgatctgc tgaaaattat caaggacaag
     4141 gacttcctgg acaatgagga aaacgaggac attctggaag atatcgtgct gaccctgaca
     4201 ctgtttgagg acagagagat gatcgaggaa cggctgaaaa cctatgccca cctgttcgac
     4261 gacaaagtga tgaagcagct gaagcggcgg agatacaccg gctggggcag gctgagccgg
     4321 aagctgatca acggcatccg ggacaagcag tccggcaaga caatcctgga tttcctgaag
     4381 tccgacggct tcgccaacag aaacttcatg cagctgatcc acgacgacag cctgaccttt
     4441 aaagaggaca tccagaaagc ccaggtgtcc ggccagggcg atagcctgca cgagcacatt
     4501 gccaatctgg ccggcagccc cgccattaag aagggcatcc tgcagacagt gaaggtggtg
     4561 gacgagctcg tgaaagtgat gggccggcac aagcccgaga acatcgtgat cgaaatggcc
     4621 agagagaacc agaccaccca gaagggacag aagaacagcc gcgagagaat gaagcggatc
     4681 gaagagggca tcaaagagct gggcagccag atcctgaaag aacaccccgt ggaaaacacc
     4741 cagctgcaga acgagaagct gtacctgtac tacctgcaga atgggcggga tatgtacgtg
     4801 gaccaggaac tggacatcaa ccggctgtcc gactacgatg tggaccatat cgtgcctcag
     4861 agctttctga aggacgactc catcgacaac aaggtgctga ccagaagcga caagaaccgg
     4921 ggcaagagcg acaacgtgcc ctccgaagag gtcgtgaaga agatgaagaa ctactggcgg
     4981 cagctgctga acgccaagct gattacccag agaaagttcg acaatctgac caaggccgag
     5041 agaggcggcc tgagcgaact ggataaggcc ggcttcatca agagacagct ggtggaaacc
     5101 cggcagatca caaagcacgt ggcacagatc ctggactccc ggatgaacac taagtacgac
     5161 gagaatgaca agctgatccg ggaagtgaaa gtgatcaccc tgaagtccaa gctggtgtcc
     5221 gatttccgga aggatttcca gttttacaaa gtgcgcgaga tcaacaacta ccaccacgcc
     5281 cacgacgcct acctgaacgc cgtcgtggga accgccctga tcaaaaagta ccctaagctg
     5341 gaaagcgagt tcgtgtacgg cgactacaag gtgtacgacg tgcggaagat gatcgccaag
     5401 agcgagcagt ctggcggctc aaaaagaacc gccgacggca gcgaattcga gcccaagaag
     5461 aagaggaaag tctaaccggt catcatcacc atcaccattg agtttaaacc cgctgatcag
     5521 cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct
     5581 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc
     5641 attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg
     5701 aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg
     5761 cggaaagaac cagctggggc tcgataccgt cgacctctag ctagagcttg gcgtaatcat
     5821 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     5881 ccggaagcat aaagtgtaaa gcctagggtg cctaatgagt gagctaactc acattaattg
     5941 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     6001 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     6061 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     6121 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     6181 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     6241 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     6301 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     6361 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     6421 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     6481 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     6541 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     6601 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     6661 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     6721 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     6781 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     6841 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     6901 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     6961 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     7021 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     7081 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     7141 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     7201 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     7261 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     7321 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     7381 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     7441 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     7501 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     7561 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     7621 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     7681 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     7741 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     7801 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     7861 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     7921 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtcg
     7981 acggatcggg agatcgatct cccgatcccc tagggtcgac tctcagtaca atctgctctg
     8041 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
     8101 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
     8161 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
     8221 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
     8281 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
     8341 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
     8401 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
     8461 tgtatc
//
