LOCUS       sequence-379989-        7917 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express TadCBEd (with eNme2-C variant) in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7917)
  AUTHORS   Neugebauer ME, Hsu A, Arbab M, Krasnow NA, McElroy AN, Pandey S, 
            Doman JL, Huang TP, Raguram A, Banskota S, Newby GA, Tolar J, Osborn
            MJ, Liu DR
  TITLE     Evolution of an adenine base editor into a small, efficient cytosine
            base editor with low off-target activity.
  JOURNAL   Nat Biotechnol. 2022 Nov 10. pii: 10.1038/s41587-022-01533-6. doi: 
            10.1038/s41587-022-01533-6.
  PUBMED    36357719
REFERENCE   2  (bases 1 to 7917)
  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. 2022 Nov 10. pii: 10.1038/s41587-022-01533-6. doi: 
            10.1038/s41587-022-01533-6."
COMMENT     Sequence Label: eNme2-C TadCBEd
FEATURES             Location/Qualifiers
     source          1..7917
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        375..578
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     529..549
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     620..639
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        620..638
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             688..708
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4576..4824
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             4855..5103
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             5146..5166
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5175..5192
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5215..5232)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5221..5445
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5516..5532)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5516..5532)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5529..5551)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5540..5556
                     /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(5564..5570,5571..5588,5589..5594))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5609..5630
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5747..5764)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5918..6506)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5998..6017)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6677..7468,7469..7537))
                     /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     7300..7319
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7538..7642)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7721..7740)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(7912..7917,1..374)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
       61 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      121 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      181 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      241 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      301 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      361 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      421 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      481 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      541 gtaggcgtgt acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc
      601 gctagagatc cgcggccgct aatacgactc actataggga gagccgccac catgaaacgg
      661 acagccgacg gaagcgagtt cgagtcacca aagaagaagc ggaaagtcag ttctgaggtg
      721 gagttttccc acgagtactg gatgagacat gccctgaccc tggccaagag ggcacgggat
      781 gagaggaagg cgcctgtggg agccgtgctg gtgctgaaca atagagtgat cggcgagggc
      841 tggaacagag ccatcggcct gcacgaccca acagcccatg ccgaaattat agccctgaga
      901 cagggcggcc tggtcatgca gaactacaga ctgattgacg ccaccctgta cgtgacattc
      961 gagccttgcg tgatgtgcgc cggcgccatg atcaactcta ggatcggccg cgtggtgttt
     1021 ggcgtgagga actcaaaaag aggcgccgca ggctccctga tgaacgtgct gaactacccc
     1081 ggaatgaatc accgcgtcga aattaccgag ggaatcctgg cagatgaatg tgccgccctg
     1141 ctgtgcgatt tctatcggat gcctagacag gtgttcaatg ctcagaagaa ggcccagagc
     1201 tccatcaact ctggcggatc tagcggagga tcctctggca gcgagacacc aggaacaagc
     1261 gagtcagcaa caccagagag cagtggcggc agcagcggcg gcagcgcagc attcaagcct
     1321 aacccaatca attacatcct gggactggca atcggaatcg catccgtggg atgggctatg
     1381 gtggagatcg acgaggaggg gaatcctatc cggctgatcg atctgggcgt gagagtgttt
     1441 gagagggccg aggtgccaaa gaccggcgat tctctggcta tggcccggag actggcacgg
     1501 agcgtgaggc gcctgacacg gagaagggca cacaggctgc tgagggcacg ccggctgctg
     1561 aagagagagg gcgtgctgca ggcagcagac ttcgatgaga atggcctgat cacgagcttg
     1621 ccaaacaccc cctggcagct gagagcagcc gccctggaca ggaagctgac accactggag
     1681 tggtctgccg tgctgctgca cctgatcaag caccgcggct acctgagcca gcggaagaac
     1741 gagggagaga cagcagccaa ggagctgggc gccctgctga agggagtggc caacaatgcc
     1801 cacgccctgc agaccggcga tttcaggaca cctgccgagc tggccctgaa taagtttgag
     1861 aaggagtccg gccacatcag aaaccagagg ggcgactata gccacacctt ctcccgcaag
     1921 gatctgcagg ccgagctgat cctgctgttc gagaagcaga aggagtttgg caatccacac
     1981 gtgagcggag gcctgaagga gggaatcgag accctgctga tgacacagag gcctgccctg
     2041 tccggcgacg cagtgcagaa gatgctgggg cactgcaccc tcgagcctac agagccaaag
     2101 gccgccaaga acacctacac agccgagcgg tttatctggc tgacaaagct gaacaatctg
     2161 agaatcctgg agcagggatc cgagaggcca ctgaccgaca cagagaggtc caccctgatg
     2221 gatgagcctt accggaagtc taaactgaca tatgcccagg ccagaaagct gctgggcctg
     2281 gaggacaccg ccttctttaa gggcctgaga tacggcaagg ataatgccga ggcctccaca
     2341 ctgatggaga tgaaggccta tcacgccatc tctcgcgccc tggagaagga gggcctgaag
     2401 gacaagaagt cccccctgaa cctgagctcc gagctgcagg atgagatcgg caccgccttc
     2461 tctctgttta agaccgacga ggatatcaca ggccgcctga aggacagggt gcagcctgag
     2521 atcctggagg ccctgctgaa gcacatctct ttcgataagt ttgtgcagat cagcctgaag
     2581 gccctgagaa ggatcgtgcc actgatggag cagggcaagc ggtacgacga ggcctgcgcc
     2641 gagatctacg gcgttcacta tggcaagaag aacacagagg agaagatcta tctgccccct
     2701 atccctgccg acgagatcag aaatcctgtg gtgctgaggg ccctgtccca ggcaagaaaa
     2761 gtgatcaacg gagtggtgcg ccggtacgga tctccagccc ggatccacat cgagaccgcc
     2821 agagaagtgg gcaagagctt caaggaccgg aaggagatcg cgaagagaca ggaggagaat
     2881 cgcaaggatc gggagaaggc cgccgccaag tttagggagt acttccctaa ctttgtgggc
     2941 gagccaaagt ctaaggacat cctgaagctg cgcctgtacg agcagcagca cggcaagtgt
     3001 ctgtatagcg gcaaagagat caatctggtg cggctgaacg agaagggcta tgtggagatc
     3061 gatcacgccc tgcctttctc cagaacctgg gacgattctt ttaacaataa ggtgctggtg
     3121 ctgggcagcg agaaccagaa taagggcaat cagacaccat acgagtattt caatggcaag
     3181 gacaactcca gggagtggca ggagttcaag gcccgcgtgg agacctctag atttcccagt
     3241 agcaagaagc agcggatcct gctgcagaag ttcgacgagg atggctttaa ggagtgcaac
     3301 ctgaatgaca ccagatacgt gaaccggttc ctgtgccagt ttgtggccga tcacatcctg
     3361 ctgaccggca agggcaagag aagggtggtc gcctctaatg gccagatcac aaacctgctg
     3421 agggggtttt ggagactgag gaaggtgcgg gcagagaatg acagacacca cgcactggat
     3481 gcagtggtgg tggcatgcag caccgtggca atgcagcaga agatcacaag attcgtgagg
     3541 tataaggaga tgaacgcctt tgacggcaag accgtcgata aggagacagg caaggtgctg
     3601 taccagaaga cccacttccc ccagccttgg gagttctttg cccaggaagt tatgatccgg
     3661 gtgttcggca agccagacgg caagcctgag tttgaggagg ccgatacccc agagaagctg
     3721 aggacactgc tggcagagaa gctgtctagc aggccagagg cagtgcacga gtacgtgacc
     3781 ccgctgttcg tgtccagggc acccaatcgg aagatgtctg gcgcccacaa ggacacactg
     3841 agaagcgcca agaggtttgt gaagcacaac gagaagatct ccgtgaagag agtgtggctg
     3901 accgagatca agctggccga tctggagaac atggtgaatt acaagaacgg cagggagatc
     3961 gagctgtatg aggccctgaa ggcaaggctg gaggcctacg gaggaaatgc caagcaggcc
     4021 ttcgacccaa aggataaccc cttttataag aagggaggac agctggtgaa ggccgtgcgg
     4081 gtggagaaga cccagaaaag cggcgtgctg ctgaataaga agaacgccta cacaatcgcc
     4141 gacaatggtg atatggtgag agtggacgtg ttctgtaagg tggataagaa gggcaagaat
     4201 cagtacttta tcgtgcctat ctatgcctgg caggtggccg agaacatcct gccagacatc
     4261 gattgcaagg gctacagaat cgacgatagc tatacattct gtttttccct gcacaagtat
     4321 gacctgatcg ccttccagaa ggatgagaag tccaaggtgg agtttgccta ctatatcaat
     4381 tgcgactcct ctagcggcgg gttctacctg gcctggcacg ataagggcag cagggagcag
     4441 cggtttcgca tctccaccca gaatctggcg ctgatccaga agtatcaggt gaacgagctg
     4501 ggcaaggaga tcaggccatg tcggctgaag aagcgcccac ccgtgagcgg cgggagcggc
     4561 gggagcgggg ggagcactaa tctgagcgac atcattgaga aggagactgg gaaacagctg
     4621 gtcattcagg agtccatcct gatgctgcct gaggaggtgg aggaagtgat cggcaacaag
     4681 ccagagtctg acatcctggt gcacaccgcc tacgacgagt ccacagatga gaatgtgatg
     4741 ctgctgacct ctgacgcccc cgagtataag ccttgggccc tggtcatcca ggattctaac
     4801 ggcgagaata agatcaagat gctgagcgga ggatccggag gatctggagg cagcaccaac
     4861 ctgtctgaca tcatcgagaa ggagacaggc aagcagctgg tcatccagga gagcatcctg
     4921 atgctgcccg aagaagtcga agaagtgatc ggaaacaagc ctgagagcga tatcctggtc
     4981 cataccgcct acgacgagag taccgacgaa aatgtgatgc tgctgacatc cgacgcccca
     5041 gagtataagc cctgggctct ggtcatccag gattccaacg gagagaacaa aatcaaaatg
     5101 ctgtctggcg gctcaaaaag aaccgccgac ggcagcgaat tcgagcccaa gaagaagagg
     5161 aaagtctaac cggtcatcat caccatcacc attgagttta aacccgctga tcagcctcga
     5221 ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc
     5281 tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc
     5341 tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt
     5401 gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa
     5461 gaaccagctg gggctcgata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat
     5521 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     5581 gcataaagtg taaagcctag ggtgcctaat gagtgagcta actcacatta attgcgttgc
     5641 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     5701 aacgcgcggg gagaggcggt ttgcgtattg ggcgcacttc cgcttcctcg ctcactgact
     5761 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     5821 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     5881 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     5941 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     6001 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     6061 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     6121 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     6181 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     6241 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     6301 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     6361 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     6421 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     6481 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgaca
     6541 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     6601 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     6661 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     6721 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     6781 gcttaccatc tggccccagt gctgcaatga taccgcggga cccacgctca ccggctccag
     6841 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     6901 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     6961 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     7021 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     7081 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     7141 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     7201 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     7261 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     7321 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     7381 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     7441 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     7501 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     7561 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     7621 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtcgacggat
     7681 cgggagatcg atctcccgat cccctagggt cgactctcag tacaatctgc tctgatgccg
     7741 catagttaag ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga
     7801 gcaaaattta agctacaaca aggcaaggct tgaccgacaa ttgcatgaag aatctgctta
     7861 gggttaggcg ttttgcgctg cttcgcgatg tacgggccag atatacgcgt tgacatt
//
