LOCUS       sequence-272345-        9024 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 9024)
  AUTHORS   Doman JL, Raguram A, Newby GA, Liu DR
  TITLE     Evaluation and minimization of Cas9-independent off-target DNA 
            editing by cytosine base editors.
  JOURNAL   Nat Biotechnol. 2020 Feb 10. pii: 10.1038/s41587-020-0414-6. doi: 
            10.1038/s41587-020-0414-6.
  PUBMED    32042165
REFERENCE   2  (bases 1 to 9024)
  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. 2020 Feb 10. pii: 10.1038/s41587-020-0414-6. doi: 
            10.1038/s41587-020-0414-6."
COMMENT     Sequence Label: YE1-BE4max-CP1028
FEATURES             Location/Qualifiers
     source          1..9024
                     /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             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             5440..5688
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             5719..5967
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             6010..6030
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6039..6056
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6079..6096)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6085..6309
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6380..6396)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6380..6396)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6393..6415)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6404..6420
                     /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(6428..6434,6435..6452,6453..6458))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6473..6494
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6611..6628)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6782..7370)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6862..6881)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7541..8332,8333..8401))
                     /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     8164..8183
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8402..8506)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8585..8604)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(8776..9024,1..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 attccccttg cggagagtgt agcagggcca tcaccgagtt cctgtccaga
      781 tatccacacg tgacactgtt tatctacatc gccaggctgt atcaccacgc agacccagag
      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 agcgagcaga gcggcgggag cggcgggagc ggggggagca ctaatctgag cgacatcatt
     5461 gagaaggaga ctgggaaaca gctggtcatt caggagtcca tcctgatgct gcctgaggag
     5521 gtggaggaag tgatcggcaa caagccagag tctgacatcc tggtgcacac cgcctacgac
     5581 gagtccacag atgaaaatgt gatgctgctg acctctgacg cccccgagta taagccttgg
     5641 gccctggtca tccaggattc taacggcgag aataagatca agatgctgag cggaggatcc
     5701 ggaggatctg gaggcagcac caacctgtct gacatcatcg agaaggagac aggcaagcag
     5761 ctggtcatcc aggagagcat cctgatgctg cccgaagaag tcgaagaagt gatcggaaac
     5821 aagcctgaga gcgatatcct ggtccatacc gcctacgacg agagtaccga cgaaaatgtg
     5881 atgctgctga catccgacgc cccagagtat aagccctggg ctctggtcat ccaggattcc
     5941 aacggagaga acaaaatcaa aatgctgtct ggcggctcaa aaagaaccgc cgacggcagc
     6001 gaattcgagc ccaagaagaa gaggaaagtc taaccggtca tcatcaccat caccattgag
     6061 tttaaacccg ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc
     6121 cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa
     6181 atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg
     6241 ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg
     6301 gctctatggc ttctgaggcg gaaagaacca gctggggctc gataccgtcg acctctagct
     6361 agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     6421 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctagggtgcc taatgagtga
     6481 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     6541 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     6601 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     6661 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     6721 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     6781 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     6841 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     6901 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     6961 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     7021 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     7081 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     7141 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     7201 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     7261 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     7321 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     7381 tgatcttttc tacggggtct gacactcagt ggaacgaaaa ctcacgttaa gggattttgg
     7441 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     7501 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     7561 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     7621 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     7681 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     7741 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     7801 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     7861 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     7921 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     7981 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     8041 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     8101 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     8161 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     8221 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     8281 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     8341 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     8401 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     8461 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     8521 aagtgccacc tgacgtcgac ggatcgggag atcgatctcc cgatccccta gggtcgactc
     8581 tcagtacaat ctgctctgat gccgcatagt taagccagta tctgctccct gcttgtgtgt
     8641 tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg
     8701 acaattgcat gaagaatctg cttagggtta ggcgttttgc gctgcttcgc gatgtacggg
     8761 ccagatatac gcgttgacat tgattattga ctagttatta atagtaatca attacggggt
     8821 cattagttca tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc
     8881 ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag
     8941 taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc
     9001 acttggcagt acatcaagtg tatc
//
