LOCUS       sequence-403578-        7827 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of N-terminally fused Nme2-rAPOBEC-nt.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7827)
  AUTHORS   Bamidele N, Zhang H, Dong X, Cheng H, Gaston N, Feinzig H, Cao H, 
            Kelly K, Watts JK, Xie J, Gao G, Sontheimer EJ
  TITLE     Domain-inlaid Nme2Cas9 adenine base editors with improved activity 
            and targeting scope.
  JOURNAL   Nat Commun. 2024 Feb 17;15(1):1458. doi: 10.1038/s41467-024-45763-5.
  PUBMED    38368418
REFERENCE   2  (bases 1 to 7827)
  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 
            Commun."; date: "2024-02-17"; volume: "15(1):1458. doi"; pages: " 
            10.1038/s41467-024-45763-5"
COMMENT     Sequence Label: pEJS2217_Nme2-rAPOBEC-nt
FEATURES             Location/Qualifiers
     source          1..7827
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        366..569
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     520..540
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     611..630
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        611..629
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             679..699
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             703..1386
                     /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             4756..5004
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             5047..5067
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5076..5093
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5116..5133)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5122..5346
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5417..5433)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5417..5433)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5430..5452)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5441..5457
                     /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(5465..5471,5472..5489,5490..5495))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5510..5531
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5648..5665)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5819..6407)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5899..5918)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6578..7369,7370..7438))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFSAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     7201..7220
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7439..7543)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7622..7641)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(7813..7827,1..365)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 ctagttatta atagtaatca attacggggt cattagttca tagcccatat atggagttcc
       61 gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat
      121 tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc
      181 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
      241 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt
      301 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
      361 ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg
      421 gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac
      481 gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg
      541 tacggtggga ggtctatata agcagagctg gtttagtgaa ccgtcagatc cgctagagat
      601 ccgcggccgc taatacgact cactataggg agagccgcca ccatgaaacg gacagccgac
      661 ggaagcgagt tcgagtcacc aaagaagaag cggaaagtca gtagctcaga gactggccca
      721 gtggctgtgg accccacatt gagacggcgg atcgagcccc atgagtttga ggtattcttc
      781 gatccgagag agctccgcaa ggagacctgc ctgctttacg aaattaattg ggggggccgg
      841 cactccattt ggcgacatac atcacagaac actaacaagc acgtcgaagt caacttcatc
      901 gagaagttca cgacagaaag atatttctgt ccgaacacaa ggtgcagcat tacctggttt
      961 ctcagctgga gcccatgcgg cgaatgtagt agggccatca ctgaattcct gtcaaggtat
     1021 ccccacgtca ctctgtttat ttacatcgca aggctgtacc accacgctga cccccgcaat
     1081 cgacaaggcc tgcgggattt gatctcttca ggtgtgacta tccaaattat gactgagcag
     1141 gagtcaggat actgctggag aaactttgtg aattatagcc cgagtaatga agcccactgg
     1201 cctaggtatc cccatctgtg ggtacgactg tacgttcttg aactgtactg catcatactg
     1261 ggcctgcctc cttgtctcaa cattctgaga aggaagcagc cacagctgac attctttacc
     1321 atcgctcttc agtcttgtca ttaccagcga ctgcccccac acattctctg ggccaccggg
     1381 ttgaaatctg gcggatctag cggaggatcc tctggcagcg agacaccagg aacaagcgag
     1441 tcagcaacac cagagagcag tggcggcagc agcggcggca gcgccgcctt caagcctaac
     1501 ccaatcaatt acatcctggg actggccatc ggaatcgcat ccgtgggatg ggctatggtg
     1561 gagatcgacg aggaggagaa tcctatccgg ctgatcgatc tgggcgtgag agtgtttgag
     1621 agggccgagg tgccaaagac cggcgattct ctggctatgg cccggagact ggcacggagc
     1681 gtgaggcgcc tgacacggag aagggcacac aggctgctga gggcacgccg gctgctgaag
     1741 agagagggcg tgctgcaggc agcagacttc gatgagaatg gcctgatcaa gagcctgcca
     1801 aacaccccct ggcagctgag agcagccgcc ctggacagga agctgacacc actggagtgg
     1861 tctgccgtgc tgctgcacct gatcaagcac cgcggctacc tgagccagcg gaagaacgag
     1921 ggagagacag cagacaagga gctgggcgcc ctgctgaagg gagtggccaa caatgcccac
     1981 gccctgcaga ccggcgattt caggacacct gccgagctgg ccctgaataa gtttgagaag
     2041 gagtccggcc acatcagaaa ccagaggggc gactatagcc acaccttctc ccgcaaggat
     2101 ctgcaggccg agctgatcct gctgttcgag aagcagaagg agtttggcaa tccacacgtg
     2161 agcggaggcc tgaaggaggg aatcgagacc ctgctgatga cacagaggcc tgccctgtcc
     2221 ggcgacgcag tgcagaagat gctgggacac tgcaccttcg agcctgcaga gccaaaggcc
     2281 gccaagaaca cctacacagc cgagcggttt atctggctga caaagctgaa caatctgaga
     2341 atcctggagc agggatccga gaggccactg accgacacag agagggccac cctgatggat
     2401 gagccttacc ggaagtctaa gctgacatat gcccaggcca gaaagctgct gggcctggag
     2461 gacaccgcct tctttaaggg cctgagatac ggcaaggata atgccgaggc ctccacactg
     2521 atggagatga aggcctatca cgccatctct cgcgccctgg agaaggaggg cctgaaggac
     2581 aagaagtccc ccctgaacct gagctccgag ctgcaggatg agatcggcac cgccttctct
     2641 ctgtttaaga ccgacgagga tatcacaggc cgcctgaagg acagggtgca gcctgagatc
     2701 ctggaggccc tgctgaagca catctctttc gataagtttg tgcagatcag cctgaaggcc
     2761 ctgagaagga tcgtgccact gatggagcag ggcaagcggt acgacgaggc ctgcgccgag
     2821 atctacggcg atcactatgg caagaagaac acagaggaga agatctatct gccccctatc
     2881 cctgccgacg agatcagaaa tcctgtggtg ctgagggccc tgtcccaggc aagaaaagtg
     2941 atcaacggag tggtgcgccg gtacggatct ccagcccgga tccacatcga gaccgccaga
     3001 gaagtgggca agagcttcaa ggaccggaag gagatcgaga agagacagga ggagaatcgc
     3061 aaggatcggg agaaggccgc cgccaagttt agggagtact tccctaactt tgtgggcgag
     3121 ccaaagtcta aggacatcct gaagctgcgc ctgtacgagc agcagcacgg caagtgtctg
     3181 tatagcggca aggagatcaa tctggtgcgg ctgaacgaga agggctatgt ggagatcgat
     3241 cacgccctgc ctttctccag aacctgggac gattctttta acaataaggt gctggtgctg
     3301 ggcagcgaga accagaataa gggcaatcag acaccatacg agtatttcaa tggcaaggac
     3361 aactccaggg agtggcagga gttcaaggcc cgcgtggaga cctctagatt tcccaggagc
     3421 aagaagcagc ggatcctgct gcagaagttc gacgaggatg gctttaagga gtgcaacctg
     3481 aatgacacca gatacgtgaa ccggttcctg tgccagtttg tggccgatca catcctgctg
     3541 accggcaagg gcaagagaag ggtgttcgcc tctaatggcc agatcacaaa cctgctgagg
     3601 ggattttggg gactgaggaa ggtgcgggca gagaatgaca gacaccacgc actggatgca
     3661 gtggtggtgg catgcagcac cgtggcaatg cagcagaaga tcacaagatt cgtgaggtat
     3721 aaggagatga acgcctttga cggcaagacc atcgataagg agacaggcaa ggtgctgcac
     3781 cagaagaccc acttccccca gccttgggag ttctttgccc aggaagtgat gatccgggtg
     3841 ttcggcaagc cagacggcaa gcctgagttt gaggaggccg ataccccaga gaagctgagg
     3901 acactgctgg cagagaagct gtctagcagg ccagaggcag tgcacgagta cgtgacccca
     3961 ctgttcgtgt ccagggcacc caatcggaag atgtctggcg cccacaagga cacactgaga
     4021 agcgccaaga ggtttgtgaa gcacaacgag aagatctccg tgaagagagt gtggctgacc
     4081 gagatcaagc tggccgatct ggagaacatg gtgaattaca agaacggcag ggagatcgag
     4141 ctgtatgagg ccctgaaggc aaggctggag gcctacggag gaaatgccaa gcaggccttc
     4201 gacccaaagg ataacccctt ttataagaag ggaggacagc tggtgaaggc cgtgcgggtg
     4261 gagaagaccc aggagagcgg cgtgctgctg aataagaaga acgcctacac aatcgccgac
     4321 aatggcgata tggtgagagt ggacgtgttc tgtaaggtgg ataagaaggg caagaatcag
     4381 tactttatcg tgcctatcta tgcctggcag gtggccgaga acatcctgcc agacatcgat
     4441 tgcaagggct acagaatcga cgatagctat acattctgtt tttccctgca caagtatgac
     4501 ctgatcgcct tccagaagga tgagaagtcc aaggtggagt ttgcctacta tatcaattgc
     4561 gactcctcta acggcaggtt ctacctggcc tggcacgata agggcagcaa ggagcagcag
     4621 tttcgcatct ccacccagaa tctggtgctg atccagaagt atcaggtgaa cgagctgggc
     4681 aaggagatca ggccatgtcg gctgaagaag cgcccacccg tgcggagcgg aggatccgga
     4741 ggatctggag gcagcaccaa cctgtctgac atcatcgaga aggagacagg caagcagctg
     4801 gtcatccagg agagcatcct gatgctgccc gaagaagtcg aagaagtgat cggaaacaag
     4861 cctgagagcg atatcctggt ccataccgcc tacgacgaga gtaccgacga aaatgtgatg
     4921 ctgctgacat ccgacgcccc agagtataag ccctgggctc tggtcatcca ggattccaac
     4981 ggagagaaca aaatcaaaat gctgtctggc ggctcaaaaa gaaccgccga cggcagcgaa
     5041 ttcgagccca agaagaagag gaaagtctaa ccggtcatca tcaccatcac cattgagttt
     5101 aaacccgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct
     5161 cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg
     5221 aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc
     5281 aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct
     5341 ctatggcttc tgaggcggaa agaaccagct ggggctcgat accgtcgacc tctagctaga
     5401 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     5461 cacacaacat acgagccgga agcataaagt gtaaagccta gggtgcctaa tgagtgagct
     5521 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     5581 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgcactt
     5641 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     5701 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     5761 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     5821 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     5881 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     5941 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     6001 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     6061 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     6121 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     6181 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     6241 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     6301 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     6361 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     6421 tcttttctac ggggtctgac actcagtgga acgaaaactc acgttaaggg attttggtca
     6481 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     6541 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     6601 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     6661 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcggg
     6721 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     6781 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     6841 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     6901 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     6961 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     7021 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     7081 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     7141 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     7201 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     7261 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     7321 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     7381 gaaggcaaaa tgccgcagaa aagggaataa gggcgacacg gaaatgttga atactcatac
     7441 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     7501 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     7561 tgccacctga cgtcgacgga tcgggagatc gatctcccga tcccctaggg tcgactctca
     7621 gtacaatctg ctctgatgcc gcatagttaa gccagtatct gctccctgct tgtgtgttgg
     7681 aggtcgctga gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc ttgaccgaca
     7741 attgcatgaa gaatctgctt agggttaggc gttttgcgct gcttcgcgat gtacgggcca
     7801 gatatacgcg ttgacattga ttattga
//
