LOCUS       sequence-226913-        8463 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 8463)
  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 8463)
  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: CP1300-CBEmax
FEATURES             Location/Qualifiers
     source          1..8463
                     /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             1513..3228
                     /codon_start=1
                     /product="N-terminal portion of Streptococcus pyogenes Cas9
                     (Zetsche et al., 2015)"
                     /label=Cas9(N)
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIE"
     CDS             5449..5469
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5478..5495
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5518..5535)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5524..5748
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5819..5835)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5819..5835)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5832..5854)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5843..5859
                     /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(5867..5873,5874..5891,5892..5897))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5912..5933
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6050..6067)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6221..6809)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6301..6320)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6980..7771,7772..7840))
                     /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     7603..7622
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7841..7945)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8024..8043)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(8215..8463,1..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 gcagcaagcc catcagagag
     1261 caggccgaga atatcatcca cctgtttacc ctgaccaatc tgggagcccc tgccgccttc
     1321 aagtactttg acaccaccat cgaccggaag aggtacacca gcaccaaaga ggtgctggac
     1381 gccaccctga tccaccagag catcaccggc ctgtacgaga cacggatcga cctgtctcag
     1441 ctgggaggcg acggcggatc aggaggctct ggcggttcag gtggatcagg cggtagcgga
     1501 ggttcaggtg gtgacaagaa gtacagcatc ggcctggcca tcggcaccaa ctctgtgggc
     1561 tgggccgtga tcaccgacga gtacaaggtg cccagcaaga aattcaaggt gctgggcaac
     1621 accgaccggc acagcatcaa gaagaacctg atcggagccc tgctgttcga cagcggcgaa
     1681 acagccgagg ccacccggct gaagagaacc gccagaagaa gatacaccag acggaagaac
     1741 cggatctgct atctgcaaga gatcttcagc aacgagatgg ccaaggtgga cgacagcttc
     1801 ttccacagac tggaagagtc cttcctggtg gaagaggata agaagcacga gcggcacccc
     1861 atcttcggca acatcgtgga cgaggtggcc taccacgaga agtaccccac catctaccac
     1921 ctgagaaaga aactggtgga cagcaccgac aaggccgacc tgcggctgat ctatctggcc
     1981 ctggcccaca tgatcaagtt ccggggccac ttcctgatcg agggcgacct gaaccccgac
     2041 aacagcgacg tggacaagct gttcatccag ctggtgcaga cctacaacca gctgttcgag
     2101 gaaaacccca tcaacgccag cggcgtggac gccaaggcca tcctgtctgc cagactgagc
     2161 aagagcagac ggctggaaaa tctgatcgcc cagctgcccg gcgagaagaa gaatggcctg
     2221 ttcggaaacc tgattgccct gagcctgggc ctgaccccca acttcaagag caacttcgac
     2281 ctggccgagg atgccaaact gcagctgagc aaggacacct acgacgacga cctggacaac
     2341 ctgctggccc agatcggcga ccagtacgcc gacctgtttc tggccgccaa gaacctgtcc
     2401 gacgccatcc tgctgagcga catcctgaga gtgaacaccg agatcaccaa ggcccccctg
     2461 agcgcctcta tgatcaagag atacgacgag caccaccagg acctgaccct gctgaaagct
     2521 ctcgtgcggc agcagctgcc tgagaagtac aaagagattt tcttcgacca gagcaagaac
     2581 ggctacgccg gctacattga cggcggagcc agccaggaag agttctacaa gttcatcaag
     2641 cccatcctgg aaaagatgga cggcaccgag gaactgctcg tgaagctgaa cagagaggac
     2701 ctgctgcgga agcagcggac cttcgacaac ggcagcatcc cccaccagat ccacctggga
     2761 gagctgcacg ccattctgcg gcggcaggaa gatttttacc cattcctgaa ggacaaccgg
     2821 gaaaagatcg agaagatcct gaccttccgc atcccctact acgtgggccc tctggccagg
     2881 ggaaacagca gattcgcctg gatgaccaga aagagcgagg aaaccatcac cccctggaac
     2941 ttcgaggaag tggtggacaa gggcgcttcc gcccagagct tcatcgagcg gatgaccaac
     3001 ttcgataaga acctgcccaa cgagaaggtg ctgcccaagc acagcctgct gtacgagtac
     3061 ttcaccgtgt ataacgagct gaccaaagtg aaatacgtga ccgagggaat gagaaagccc
     3121 gccttcctga gcggcgagca gaaaaaggcc atcgtggacc tgctgttcaa gaccaaccgg
     3181 aaagtgaccg tgaagcagct gaaagaggac tacttcaaga aaatcgagtg cttcgactcc
     3241 gtggaaatct ccggcgtgga agatcggttc aacgcctccc tgggcacata ccacgatctg
     3301 ctgaaaatta tcaaggacaa ggacttcctg gacaatgagg aaaacgagga cattctggaa
     3361 gatatcgtgc tgaccctgac actgtttgag gacagagaga tgatcgagga acggctgaaa
     3421 acctatgccc acctgttcga cgacaaagtg atgaagcagc tgaagcggcg gagatacacc
     3481 ggctggggca ggctgagccg gaagctgatc aacggcatcc gggacaagca gtccggcaag
     3541 acaatcctgg atttcctgaa gtccgacggc ttcgccaaca gaaacttcat gcagctgatc
     3601 cacgacgaca gcctgacctt taaagaggac atccagaaag cccaggtgtc cggccagggc
     3661 gatagcctgc acgagcacat tgccaatctg gccggcagcc ccgccattaa gaagggcatc
     3721 ctgcagacag tgaaggtggt ggacgagctc gtgaaagtga tgggccggca caagcccgag
     3781 aacatcgtga tcgaaatggc cagagagaac cagaccaccc agaagggaca gaagaacagc
     3841 cgcgagagaa tgaagcggat cgaagagggc atcaaagagc tgggcagcca gatcctgaaa
     3901 gaacaccccg tggaaaacac ccagctgcag aacgagaagc tgtacctgta ctacctgcag
     3961 aatgggcggg atatgtacgt ggaccaggaa ctggacatca accggctgtc cgactacgat
     4021 gtggaccata tcgtgcctca gagctttctg aaggacgact ccatcgacaa caaggtgctg
     4081 accagaagcg acaagaaccg gggcaagagc gacaacgtgc cctccgaaga ggtcgtgaag
     4141 aagatgaaga actactggcg gcagctgctg aacgccaagc tgattaccca gagaaagttc
     4201 gacaatctga ccaaggccga gagaggcggc ctgagcgaac tggataaggc cggcttcatc
     4261 aagagacagc tggtggaaac ccggcagatc acaaagcacg tggcacagat cctggactcc
     4321 cggatgaaca ctaagtacga cgagaatgac aagctgatcc gggaagtgaa agtgatcacc
     4381 ctgaagtcca agctggtgtc cgatttccgg aaggatttcc agttttacaa agtgcgcgag
     4441 atcaacaact accaccacgc ccacgacgcc tacctgaacg ccgtcgtggg aaccgccctg
     4501 atcaaaaagt accctaagct ggaaagcgag ttcgtgtacg gcgactacaa ggtgtacgac
     4561 gtgcggaaga tgatcgccaa gagcgagcag gaaatcggca aggctaccgc caagtacttc
     4621 ttctacagca acatcatgaa ctttttcaag accgagatta ccctggccaa cggcgagatc
     4681 cggaagcggc ctctgatcga gacaaacggc gaaaccgggg agatcgtgtg ggataagggc
     4741 cgggattttg ccaccgtgcg gaaagtgctg agcatgcccc aagtgaatat cgtgaaaaag
     4801 accgaggtgc agacaggcgg cttcagcaaa gagtctatcc tgcccaagag gaacagcgat
     4861 aagctgatcg ccagaaagaa ggactgggac cctaagaagt acggcggctt cgacagcccc
     4921 accgtggcct attctgtgct ggtggtggcc aaagtggaaa agggcaagtc caagaaactg
     4981 aagagtgtga aagagctgct ggggatcacc atcatggaaa gaagcagctt cgagaagaat
     5041 cccatcgact ttctggaagc caagggctac aaagaagtga aaaaggacct gatcatcaag
     5101 ctgcctaagt actccctgtt cgagctggaa aacggccgga agagaatgct ggcctctgcc
     5161 ggcgaactgc agaagggaaa cgaactggcc ctgccctcca aatatgtgaa cttcctgtac
     5221 ctggccagcc actatgagaa gctgaagggc tcccccgagg ataatgagca gaaacagctg
     5281 tttgtggaac agcacaagca ctacctggac gagatcatcg agcagatcag cgagttctcc
     5341 aagagagtga tcctggccga cgctaatctg gacaaagtgc tgtccgccta caacaagcac
     5401 cgggattctg gcggctcaaa aagaaccgcc gacggcagcg aattcgagcc caagaagaag
     5461 aggaaagtct aaccggtcat catcaccatc accattgagt ttaaacccgc tgatcagcct
     5521 cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga
     5581 ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt
     5641 gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg
     5701 attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg
     5761 aaagaaccag ctggggctcg ataccgtcga cctctagcta gagcttggcg taatcatggt
     5821 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     5881 gaagcataaa gtgtaaagcc tagggtgcct aatgagtgag ctaactcaca ttaattgcgt
     5941 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     6001 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     6061 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     6121 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     6181 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     6241 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     6301 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     6361 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     6421 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     6481 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     6541 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     6601 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     6661 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     6721 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     6781 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     6841 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     6901 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     6961 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     7021 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     7081 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     7141 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     7201 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     7261 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     7321 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     7381 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     7441 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     7501 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     7561 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     7621 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     7681 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     7741 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     7801 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     7861 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     7921 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg
     7981 gatcgggaga tcgatctccc gatcccctag ggtcgactct cagtacaatc tgctctgatg
     8041 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
     8101 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
     8161 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
     8221 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
     8281 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
     8341 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
     8401 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
     8461 atc
//
