LOCUS       sequence-379991-        7917 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express TadDE (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 TadDE
FEATURES             Location/Qualifiers
     source          1..7917
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        367..570
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     521..541
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     612..631
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        612..630
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             680..700
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4568..4816
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             4847..5095
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             5138..5158
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5167..5184
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5207..5224)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5213..5437
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5508..5524)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5508..5524)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5521..5543)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5532..5548
                     /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(5556..5562,5563..5580,5581..5586))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5601..5622
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5739..5756)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5910..6498)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5990..6009)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6669..7460,7461..7529))
                     /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     7292..7311
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7530..7634)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7713..7732)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(7904..7917,1..366)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 actagttatt aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc
       61 cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca
      121 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt
      181 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg
      241 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag
      301 tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt
      361 accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg
      421 ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa
      481 cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt
      541 gtacggtggg aggtctatat aagcagagct ggtttagtga accgtcagat ccgctagaga
      601 tccgcggccg ctaatacgac tcactatagg gagagccgcc accatgaaac ggacagccga
      661 cggaagcgag ttcgagtcac caaagaagaa gcggaaagtc agttctgagg tggagttttc
      721 ccacgagtac tggatgagac atgccctgac cctggccaag agggcacggg atgagggaga
      781 ggcgcctgtg ggagccgtgc tggtgctgaa caatagagtg atcggcgagg gctggaacag
      841 acgtatcggc ctgcacgacc caacagccca tgccgaaatt atggccctga gacagggcgg
      901 cctggtcatg cagaactcca gactgattga cgccaccctg tacgtgacat tcgagccttg
      961 cgtgatgtgc gccggcgcca tgatcaactc taggatcggc cgcgtggtgt ttggcgtgag
     1021 gaactcaaaa agaggcgccg caggctccct gatgaacgtg ctgaactacc ccggcatgaa
     1081 tcaccgcgtc gaaattaccg agggaatcct ggcagatgaa tgtgccgccc tgctgtgcga
     1141 tttctatcgg atgcctagac aggtgttcaa tgctcagaag aaggcccaga gctccatcaa
     1201 ctctggcgga tctagcggag gatcctctgg cagcgagaca ccaggaacaa gcgagtcagc
     1261 aacaccagag agcagtggcg gcagcagcgg cggcagcgca gcattcaagc ctaacccaat
     1321 caattacatc ctgggactgg caatcggaat cgcatccgtg ggatgggcta tggtggagat
     1381 cgacgaggag gggaatccta tccggctgat cgatctgggc gtgagagtgt ttgagagggc
     1441 cgaggtgcca aagaccggcg attctctggc tatggcccgg agactggcac ggagcgtgag
     1501 gcgcctgaca cggagaaggg cacacaggct gctgagggca cgccggctgc tgaagagaga
     1561 gggcgtgctg caggcagcag acttcgatga gaatggcctg atcacgagct tgccaaacac
     1621 cccctggcag ctgagagcag ccgccctgga caggaagctg acaccactgg agtggtctgc
     1681 cgtgctgctg cacctgatca agcaccgcgg ctacctgagc cagcggaaga acgagggaga
     1741 gacagcagcc aaggagctgg gcgccctgct gaagggagtg gccaacaatg cccacgccct
     1801 gcagaccggc gatttcagga cacctgccga gctggccctg aataagtttg agaaggagtc
     1861 cggccacatc agaaaccaga ggggcgacta tagccacacc ttctcccgca aggatctgca
     1921 ggccgagctg atcctgctgt tcgagaagca gaaggagttt ggcaatccac acgtgagcgg
     1981 aggcctgaag gagggaatcg agaccctgct gatgacacag aggcctgccc tgtccggcga
     2041 cgcagtgcag aagatgctgg ggcactgcac cctcgagcct acagagccaa aggccgccaa
     2101 gaacacctac acagccgagc ggtttatctg gctgacaaag ctgaacaatc tgagaatcct
     2161 ggagcaggga tccgagaggc cactgaccga cacagagagg tccaccctga tggatgagcc
     2221 ttaccggaag tctaaactga catatgccca ggccagaaag ctgctgggcc tggaggacac
     2281 cgccttcttt aagggcctga gatacggcaa ggataatgcc gaggcctcca cactgatgga
     2341 gatgaaggcc tatcacgcca tctctcgcgc cctggagaag gagggcctga aggacaagaa
     2401 gtcccccctg aacctgagct ccgagctgca ggatgagatc ggcaccgcct tctctctgtt
     2461 taagaccgac gaggatatca caggccgcct gaaggacagg gtgcagcctg agatcctgga
     2521 ggccctgctg aagcacatct ctttcgataa gtttgtgcag atcagcctga aggccctgag
     2581 aaggatcgtg ccactgatgg agcagggcaa gcggtacgac gaggcctgcg ccgagatcta
     2641 cggcgttcac tatggcaaga agaacacaga ggagaagatc tatctgcccc ctatccctgc
     2701 cgacgagatc agaaatcctg tggtgctgag ggccctgtcc caggcaagaa aagtgatcaa
     2761 cggagtggtg cgccggtacg gatctccagc ccggatccac atcgagaccg ccagagaagt
     2821 gggcaagagc ttcaaggacc ggaaggagat cgcgaagaga caggaggaga atcgcaagga
     2881 tcgggagaag gccgccgcca agtttaggga gtacttccct aactttgtgg gcgagccaaa
     2941 gtctaaggac atcctgaagc tgcgcctgta cgagcagcag cacggcaagt gtctgtatag
     3001 cggcaaagag atcaatctgg tgcggctgaa cgagaagggc tatgtggaga tcgatcacgc
     3061 cctgcctttc tccagaacct gggacgattc ttttaacaat aaggtgctgg tgctgggcag
     3121 cgagaaccag aataagggca atcagacacc atacgagtat ttcaatggca aggacaactc
     3181 cagggagtgg caggagttca aggcccgcgt ggagacctct agatttccca gtagcaagaa
     3241 gcagcggatc ctgctgcaga agttcgacga ggatggcttt aaggagtgca acctgaatga
     3301 caccagatac gtgaaccggt tcctgtgcca gtttgtggcc gatcacatcc tgctgaccgg
     3361 caagggcaag agaagggtgg tcgcctctaa tggccagatc acaaacctgc tgagggggtt
     3421 ttggagactg aggaaggtgc gggcagagaa tgacagacac cacgcactgg atgcagtggt
     3481 ggtggcatgc agcaccgtgg caatgcagca gaagatcaca agattcgtga ggtataagga
     3541 gatgaacgcc tttgacggca agaccgtcga taaggagaca ggcaaggtgc tgtaccagaa
     3601 gacccacttc ccccagcctt gggagttctt tgcccaggaa gttatgatcc gggtgttcgg
     3661 caagccagac ggcaagcctg agtttgagga ggccgatacc ccagagaagc tgaggacact
     3721 gctggcagag aagctgtcta gcaggccaga ggcagtgcac gagtacgtga ccccgctgtt
     3781 cgtgtccagg gcacccaatc ggaagatgtc tggcgcccac aaggacacac tgagaagcgc
     3841 caagaggttt gtgaagcaca acgagaagat ctccgtgaag agagtgtggc tgaccgagat
     3901 caagctggcc gatctggaga acatggtgaa ttacaagaac ggcagggaga tcgagctgta
     3961 tgaggccctg aaggcaaggc tggaggccta cggaggaaat gccaagcagg ccttcgaccc
     4021 aaaggataac cccttttata agaagggagg acagctggtg aaggccgtgc gggtggagaa
     4081 gacccagaaa agcggcgtgc tgctgaataa gaagaacgcc tacacaatcg ccgacaatgg
     4141 tgatatggtg agagtggacg tgttctgtaa ggtggataag aagggcaaga atcagtactt
     4201 tatcgtgcct atctatgcct ggcaggtggc cgagaacatc ctgccagaca tcgattgcaa
     4261 gggctacaga atcgacgata gctatacatt ctgtttttcc ctgcacaagt atgacctgat
     4321 cgccttccag aaggatgaga agtccaaggt ggagtttgcc tactatatca attgcgactc
     4381 ctctagcggc gggttctacc tggcctggca cgataagggc agcagggagc agcggtttcg
     4441 catctccacc cagaatctgg cgctgatcca gaagtatcag gtgaacgagc tgggcaagga
     4501 gatcaggcca tgtcggctga agaagcgccc acccgtgagc ggcgggagcg gcgggagcgg
     4561 ggggagcact aatctgagcg acatcattga gaaggagact gggaaacagc tggtcattca
     4621 ggagtccatc ctgatgctgc ctgaggaggt ggaggaagtg atcggcaaca agccagagtc
     4681 tgacatcctg gtgcacaccg cctacgacga gtccacagat gagaatgtga tgctgctgac
     4741 ctctgacgcc cccgagtata agccttgggc cctggtcatc caggattcta acggcgagaa
     4801 taagatcaag atgctgagcg gaggatccgg aggatctgga ggcagcacca acctgtctga
     4861 catcatcgag aaggagacag gcaagcagct ggtcatccag gagagcatcc tgatgctgcc
     4921 cgaagaagtc gaagaagtga tcggaaacaa gcctgagagc gatatcctgg tccataccgc
     4981 ctacgacgag agtaccgacg aaaatgtgat gctgctgaca tccgacgccc cagagtataa
     5041 gccctgggct ctggtcatcc aggattccaa cggagagaac aaaatcaaaa tgctgtctgg
     5101 cggctcaaaa agaaccgccg acggcagcga attcgagccc aagaagaaga ggaaagtcta
     5161 accggtcatc atcaccatca ccattgagtt taaacccgct gatcagcctc gactgtgcct
     5221 tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt
     5281 gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg
     5341 tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac
     5401 aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc
     5461 tggggctcga taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt
     5521 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     5581 tgtaaagcct agggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     5641 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     5701 gggagaggcg gtttgcgtat tgggcgcact tccgcttcct cgctcactga ctcgctgcgc
     5761 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     5821 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     5881 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     5941 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     6001 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     6061 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     6121 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     6181 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     6241 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     6301 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     6361 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     6421 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     6481 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cactcagtgg
     6541 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     6601 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     6661 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     6721 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     6781 tctggcccca gtgctgcaat gataccgcgg gacccacgct caccggctcc agatttatca
     6841 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     6901 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     6961 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     7021 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     7081 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     7141 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     7201 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     7261 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     7321 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     7381 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     7441 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     7501 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     7561 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     7621 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcgggagat
     7681 cgatctcccg atcccctagg gtcgactctc agtacaatct gctctgatgc cgcatagtta
     7741 agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt
     7801 taagctacaa caaggcaagg cttgaccgac aattgcatga agaatctgct tagggttagg
     7861 cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc gttgacattg attattg
//
