LOCUS       sequence-379988-        7917 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express TadCBEa (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 TadCBEa
FEATURES             Location/Qualifiers
     source          1..7917
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        376..579
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     530..550
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     621..640
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        621..639
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             689..709
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4577..4825
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             4856..5104
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             5147..5167
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5176..5193
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5216..5233)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5222..5446
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5517..5533)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5517..5533)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5530..5552)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5541..5557
                     /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(5565..5571,5572..5589,5590..5595))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5610..5631
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5748..5765)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5919..6507)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5999..6018)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6678..7469,7470..7538))
                     /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     7301..7320
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7539..7643)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7722..7741)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(7913..7917,1..375)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 tgattattga ctagttatta atagtaatca attacggggt cattagttca tagcccatat
       61 atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac
      121 ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc
      181 cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg
      241 tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat
      301 tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc
      361 atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt
      421 gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac
      481 caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc
      541 ggtaggcgtg tacggtggga ggtctatata agcagagctg gtttagtgaa ccgtcagatc
      601 cgctagagat ccgcggccgc taatacgact cactataggg agagccgcca ccatgaaacg
      661 gacagccgac ggaagcgagt tcgagtcacc aaagaagaag cggaaagtca gttctgaggt
      721 ggagttttcc cacgagtact ggatgagaca tgccctgacc ctggccaaga gggcacggga
      781 tgagggggcg ggtcctgtgg gagccgtgct ggtgctgaac aatagagtga tcggcgaggg
      841 ctggaacaga gccatcggcc tgcacgaccc aacagcccat gccgaaatta tggccctgag
      901 acagggcggc ctggtcatgc agaactacag actgtttgac gccaccctgt acgtgacatt
      961 cgagccttgc gtgatgtgcg ccggcgccat gatcaactct aggatcggcc gcgtggtgtt
     1021 tggcgtgagg aactcaaaaa gaggcgccgc aggctccctg atgaacgtgc tgaactaccc
     1081 cggcatgaat caccgcgtcg aaattaccga gggaatcctg gcagatgaat gtgccgccct
     1141 gctgtgcgat ttctatcgga tgcctagaca ggtgttcaat tctcagaaga aggcccagag
     1201 ctccatcaac tctggcggat ctagcggagg atcctctggc agcgagacac caggaacaag
     1261 cgagtcagca acaccagaga gcagtggcgg cagcagcggc ggcagcgcag cattcaagcc
     1321 taacccaatc aattacatcc tgggactggc aatcggaatc gcatccgtgg gatgggctat
     1381 ggtggagatc gacgaggagg ggaatcctat ccggctgatc gatctgggcg tgagagtgtt
     1441 tgagagggcc gaggtgccaa agaccggcga ttctctggct atggcccgga gactggcacg
     1501 gagcgtgagg cgcctgacac ggagaagggc acacaggctg ctgagggcac gccggctgct
     1561 gaagagagag ggcgtgctgc aggcagcaga cttcgatgag aatggcctga tcacgagctt
     1621 gccaaacacc ccctggcagc tgagagcagc cgccctggac aggaagctga caccactgga
     1681 gtggtctgcc gtgctgctgc acctgatcaa gcaccgcggc tacctgagcc agcggaagaa
     1741 cgagggagag acagcagcca aggagctggg cgccctgctg aagggagtgg ccaacaatgc
     1801 ccacgccctg cagaccggcg atttcaggac acctgccgag ctggccctga ataagtttga
     1861 gaaggagtcc ggccacatca gaaaccagag gggcgactat agccacacct tctcccgcaa
     1921 ggatctgcag gccgagctga tcctgctgtt cgagaagcag aaggagtttg gcaatccaca
     1981 cgtgagcgga ggcctgaagg agggaatcga gaccctgctg atgacacaga ggcctgccct
     2041 gtccggcgac gcagtgcaga agatgctggg gcactgcacc ctcgagccta cagagccaaa
     2101 ggccgccaag aacacctaca cagccgagcg gtttatctgg ctgacaaagc tgaacaatct
     2161 gagaatcctg gagcagggat ccgagaggcc actgaccgac acagagaggt ccaccctgat
     2221 ggatgagcct taccggaagt ctaaactgac atatgcccag gccagaaagc tgctgggcct
     2281 ggaggacacc gccttcttta agggcctgag atacggcaag gataatgccg aggcctccac
     2341 actgatggag atgaaggcct atcacgccat ctctcgcgcc ctggagaagg agggcctgaa
     2401 ggacaagaag tcccccctga acctgagctc cgagctgcag gatgagatcg gcaccgcctt
     2461 ctctctgttt aagaccgacg aggatatcac aggccgcctg aaggacaggg tgcagcctga
     2521 gatcctggag gccctgctga agcacatctc tttcgataag tttgtgcaga tcagcctgaa
     2581 ggccctgaga aggatcgtgc cactgatgga gcagggcaag cggtacgacg aggcctgcgc
     2641 cgagatctac ggcgttcact atggcaagaa gaacacagag gagaagatct atctgccccc
     2701 tatccctgcc gacgagatca gaaatcctgt ggtgctgagg gccctgtccc aggcaagaaa
     2761 agtgatcaac ggagtggtgc gccggtacgg atctccagcc cggatccaca tcgagaccgc
     2821 cagagaagtg ggcaagagct tcaaggaccg gaaggagatc gcgaagagac aggaggagaa
     2881 tcgcaaggat cgggagaagg ccgccgccaa gtttagggag tacttcccta actttgtggg
     2941 cgagccaaag tctaaggaca tcctgaagct gcgcctgtac gagcagcagc acggcaagtg
     3001 tctgtatagc ggcaaagaga tcaatctggt gcggctgaac gagaagggct atgtggagat
     3061 cgatcacgcc ctgcctttct ccagaacctg ggacgattct tttaacaata aggtgctggt
     3121 gctgggcagc gagaaccaga ataagggcaa tcagacacca tacgagtatt tcaatggcaa
     3181 ggacaactcc agggagtggc aggagttcaa ggcccgcgtg gagacctcta gatttcccag
     3241 tagcaagaag cagcggatcc tgctgcagaa gttcgacgag gatggcttta aggagtgcaa
     3301 cctgaatgac accagatacg tgaaccggtt cctgtgccag tttgtggccg atcacatcct
     3361 gctgaccggc aagggcaaga gaagggtggt cgcctctaat ggccagatca caaacctgct
     3421 gagggggttt tggagactga ggaaggtgcg ggcagagaat gacagacacc acgcactgga
     3481 tgcagtggtg gtggcatgca gcaccgtggc aatgcagcag aagatcacaa gattcgtgag
     3541 gtataaggag atgaacgcct ttgacggcaa gaccgtcgat aaggagacag gcaaggtgct
     3601 gtaccagaag acccacttcc cccagccttg ggagttcttt gcccaggaag ttatgatccg
     3661 ggtgttcggc aagccagacg gcaagcctga gtttgaggag gccgataccc cagagaagct
     3721 gaggacactg ctggcagaga agctgtctag caggccagag gcagtgcacg agtacgtgac
     3781 cccgctgttc gtgtccaggg cacccaatcg gaagatgtct ggcgcccaca aggacacact
     3841 gagaagcgcc aagaggtttg tgaagcacaa cgagaagatc tccgtgaaga gagtgtggct
     3901 gaccgagatc aagctggccg atctggagaa catggtgaat tacaagaacg gcagggagat
     3961 cgagctgtat gaggccctga aggcaaggct ggaggcctac ggaggaaatg ccaagcaggc
     4021 cttcgaccca aaggataacc ccttttataa gaagggagga cagctggtga aggccgtgcg
     4081 ggtggagaag acccagaaaa gcggcgtgct gctgaataag aagaacgcct acacaatcgc
     4141 cgacaatggt gatatggtga gagtggacgt gttctgtaag gtggataaga agggcaagaa
     4201 tcagtacttt atcgtgccta tctatgcctg gcaggtggcc gagaacatcc tgccagacat
     4261 cgattgcaag ggctacagaa tcgacgatag ctatacattc tgtttttccc tgcacaagta
     4321 tgacctgatc gccttccaga aggatgagaa gtccaaggtg gagtttgcct actatatcaa
     4381 ttgcgactcc tctagcggcg ggttctacct ggcctggcac gataagggca gcagggagca
     4441 gcggtttcgc atctccaccc agaatctggc gctgatccag aagtatcagg tgaacgagct
     4501 gggcaaggag atcaggccat gtcggctgaa gaagcgccca cccgtgagcg gcgggagcgg
     4561 cgggagcggg gggagcacta atctgagcga catcattgag aaggagactg ggaaacagct
     4621 ggtcattcag gagtccatcc tgatgctgcc tgaggaggtg gaggaagtga tcggcaacaa
     4681 gccagagtct gacatcctgg tgcacaccgc ctacgacgag tccacagatg agaatgtgat
     4741 gctgctgacc tctgacgccc ccgagtataa gccttgggcc ctggtcatcc aggattctaa
     4801 cggcgagaat aagatcaaga tgctgagcgg aggatccgga ggatctggag gcagcaccaa
     4861 cctgtctgac atcatcgaga aggagacagg caagcagctg gtcatccagg agagcatcct
     4921 gatgctgccc gaagaagtcg aagaagtgat cggaaacaag cctgagagcg atatcctggt
     4981 ccataccgcc tacgacgaga gtaccgacga aaatgtgatg ctgctgacat ccgacgcccc
     5041 agagtataag ccctgggctc tggtcatcca ggattccaac ggagagaaca aaatcaaaat
     5101 gctgtctggc ggctcaaaaa gaaccgccga cggcagcgaa ttcgagccca agaagaagag
     5161 gaaagtctaa ccggtcatca tcaccatcac cattgagttt aaacccgctg atcagcctcg
     5221 actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc
     5281 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt
     5341 ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat
     5401 tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa
     5461 agaaccagct ggggctcgat accgtcgacc tctagctaga gcttggcgta atcatggtca
     5521 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     5581 agcataaagt gtaaagccta gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     5641 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     5701 caacgcgcgg ggagaggcgg tttgcgtatt gggcgcactt ccgcttcctc gctcactgac
     5761 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     5821 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     5881 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     5941 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     6001 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     6061 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     6121 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     6181 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     6241 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     6301 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     6361 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     6421 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     6481 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     6541 actcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     6601 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     6661 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     6721 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     6781 ggcttaccat ctggccccag tgctgcaatg ataccgcggg acccacgctc accggctcca
     6841 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     6901 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     6961 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     7021 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     7081 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     7141 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     7201 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     7261 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     7321 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     7381 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     7441 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     7501 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     7561 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     7621 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtcgacgga
     7681 tcgggagatc gatctcccga tcccctaggg tcgactctca gtacaatctg ctctgatgcc
     7741 gcatagttaa gccagtatct gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg
     7801 agcaaaattt aagctacaac aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt
     7861 agggttaggc gttttgcgct gcttcgcgat gtacgggcca gatatacgcg ttgacat
//
