LOCUS       sequence-419195-        7410 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of domain inlaid Nme2.Smu-ABE8e-i7.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7410)
  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 7410)
  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: pEJS2423_Nme2.Smu-ABE8e-i7
FEATURES             Location/Qualifiers
     source          1..7410
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        2..381
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        382..585
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     536..556
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     627..646
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        627..645
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             695..715
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      4184..4193
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4646..4666
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4675..4692
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4715..4732)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4721..4945
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5016..5032)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5016..5032)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5029..5051)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5040..5056
                     /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(5064..5070,5071..5088,5089..5094))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5109..5130
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5247..5264)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5418..6006)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5498..5517)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6177..6968,6969..7037))
                     /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     6800..6819
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7038..7142)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7221..7240)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tgacattgat tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc
       61 ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc
      121 aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg
      181 actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat
      241 caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc
      301 tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta
      361 ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag
      421 cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt
      481 tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa
      541 atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt
      601 cagatccgct agagatccgc ggccgctaat acgactcact atagggagag ccgccaccat
      661 gaaacggaca gccgacggaa gcgagttcga gtcaccaaag aagaagcgga aagtcgaaga
      721 tatggccgcc ttcaagccta acccaatcaa ttacatcctg ggactggcca tcggaatcgc
      781 atccgtggga tgggctatgg tggagatcga cgaggaggag aatcctatcc ggctgatcga
      841 tctgggcgtg agagtgtttg agagggccga ggtgccaaag accggcgatt ctctggctat
      901 ggcccggaga ctggcacgga gcgtgaggcg cctgacacgg agaagggcac acaggctgct
      961 gagggcacgc cggctgctga agagagaggg cgtgctgcag gcagcagact tcgatgagaa
     1021 tggcctgatc aagagcctgc caaacacccc ctggcagctg agagcagccg ccctggacag
     1081 gaagctgaca ccactggagt ggtctgccgt gctgctgcac ctgatcaagc accgcggcta
     1141 cctgagccag cggaagaacg agggagagac agcagacaag gagctgggcg ccctgctgaa
     1201 gggagtggcc aacaatgccc acgccctgca gaccggcgat ttcaggacac ctgccgagct
     1261 ggccctgaat aagtttgaga aggagtccgg ccacatcaga aaccagaggg gcgactatag
     1321 ccacaccttc tcccgcaagg atctgcaggc cgagctgatc ctgctgttcg agaagcagaa
     1381 ggagtttggc aatccacacg tgagcggagg cctgaaggag ggaatcgaga ccctgctgat
     1441 gacacagagg cctgccctgt ccggcgacgc agtgcagaag atgctgggac actgcacctt
     1501 cgagcctgca gagccaaagg ccgccaagaa cacctacaca gccgagcggt ttatctggct
     1561 gacaaagctg aacaatctga gaatcctgga gcagggatcc gagaggccac tgaccgacac
     1621 agagagggcc accctgatgg atgagcctta ccggaagtct aagctgacat atgcccaggc
     1681 cagaaagctg ctgggcctgg aggacaccgc cttctttaag ggcctgagat acggcaagga
     1741 taatgccgag gcctccacac tgatggagat gaaggcctat cacgccatct ctcgcgccct
     1801 ggagaaggag ggcctgaagg acaagaagtc ccccctgaac ctgagctccg agctgcagga
     1861 tgagatcggc accgccttct ctctgtttaa gaccgacgag gatatcacag gccgcctgaa
     1921 ggacagggtg cagcctgaga tcctggaggc cctgctgaag cacatctctt tcgataagtt
     1981 tgtgcagatc agcctgaagg ccctgagaag gatcgtgcca ctgatggagc agggcaagcg
     2041 gtacgacgag gcctgcgccg agatctacgg cgatcactat ggcaagaaga acacagagga
     2101 gaagatctat ctgcccccta tccctgccga cgagatcaga aatcctgtgg tgctgagggc
     2161 cctgtcccag gcaagaaaag tgatcaacgg agtggtgcgc cggtacggat ctccagcccg
     2221 gatccacatc gagaccgcca gagaagtggg caagagcttc aaggaccgga aggagatcga
     2281 gaagagacag gaggagaatc gcaaggatcg ggagaaggcc gccgccaagt ttagggagta
     2341 cttccctaac tttgtgggcg agccaaagtc taaggacatc ctgaagctgc gcctgtacga
     2401 gcagcagcac ggcaagtgtc tgtatagcgg caaggagatc aatctggtgc ggctgaacga
     2461 gaagggctat gtggagatcg atcacgccct gcctttctcc agaacctggg acgattcttt
     2521 taacaataag gtgctggtgc tgggcagcga gaaccagaat aagggcaatc agacaccata
     2581 cgagtatttc aatggcaagg acaactccag ggagtggcag gagttcaagg cccgcgtgga
     2641 gacctctaga tttcccagga gcaagaagca gcggatcctg ctgcagaagt tcgacgagga
     2701 tggctttaag gagtgcaacc tgaatgacac cagatacgtg aaccggttcc tgtgccagtt
     2761 tgtggccgat cacatcctgc tgaccggcaa gggcaagaga agggtgttcg cctctaatgg
     2821 ccagatcaca aacctgctga ggggattttg gggactgagg aaggtgcggg cagagaatga
     2881 cagacaccac gcactggatg cagtggtggt ggcatgcagc accgtggcaa tgcagcagaa
     2941 gatcacaaga ttcgtgaggt ataaggagat gaacgccttt gacggcaaga ccatcgataa
     3001 ggagggcgga tcaggaggct ctggcggttc aggtggatca ggcggtagcg gaggttcagg
     3061 tggttctgag gtggagtttt cccacgagta ctggatgaga catgccctga ccctggccaa
     3121 gagggcacgc gatgagaggg aggtgcctgt gggagccgtg ctggtgctga acaatagagt
     3181 gatcggcgag ggctggaaca gagccatcgg cctgcacgac ccaacagccc atgccgaaat
     3241 tatggccctg agacagggcg gcctggtcat gcagaactac agactgattg acgccaccct
     3301 gtacgtgaca ttcgagcctt gcgtgatgtg cgccggcgcc atgatccact ctaggatcgg
     3361 ccgcgtggtg tttggcgtga ggaacagcaa acggggcgcc gcaggctccc tgatgaacgt
     3421 gctgaactac cccggcatga atcaccgcgt cgaaattacc gagggaatcc tggcagatga
     3481 atgtgccgcc ctgctgtgcg acttctaccg gatgcctaga caggtgttca atgctcagaa
     3541 gaaggcccag agctccatca acggctcctc tggctctgag acacctggca caagcgagag
     3601 cgcaacacct gaaagcagcg gcacaggcaa ggtgctgcac cagaagaccc acttccccca
     3661 gccttgggag ttctttgccc aggaagtgat gatccgggtg ttcggcaagc cagacggcaa
     3721 gcctgagttt gaggaggccg ataccccaga gaagctgagg acactgctgg cagagaagct
     3781 gtctagcagg ccagaggcag tgcacgagta cgtgacccca ctgttcgtgt ccagggcacc
     3841 caatcggaag atgtctggcg cccacaagga cacactgaga agcgccaaga ggtttgtgaa
     3901 gcacaacgag aagatctccg tgaagagagt gtggctgacc gagatcaagc tggccgatct
     3961 ggagaacatg gtgaattaca agaacggcag ggagatcgag ctgtatgagg ccctgaaggc
     4021 aaggctggag gcctacggag gaaatgccaa gcaggccttc gacccaaagg ataacccctt
     4081 ttataagaag ggaggacagc tggtgaaggc cgtgcgggtg gagaagaccc aggagagcgg
     4141 cgtgctgctg aataagaaga acgcctacac aatcgccgac aacgccacca tggtgcgggt
     4201 ggacgtgtac accaaggccg gcaagaacta cctggttcct gtgtacgtgt ggcaggtggc
     4261 ccagggcatc ttacccaacc gcgccgtgac cagcggcaag tccgaggctg actgggacct
     4321 gatcgatgag agcttcgagt tcaagttctc tctgtcccgg ggagatctcg tggaaatgat
     4381 ctccaacaag ggcagaatct tcggctacta caacggcctg gacagagcca acggctctat
     4441 tggaattaga gagcacgacc tagagaagag caagggcaaa gacggcgtgc atagagtggg
     4501 agtgaaaaca gctacagcat ttaacaagta ccacgtggat cccctgggca aagagatcca
     4561 cagatgcagc agcgaaccca gacctacact gaaaatcaag tctaagaagg aggataaaag
     4621 aaccgccgac ggcagcgaat tcgagcccaa gaagaagagg aaagtctaac cggtcatcat
     4681 caccatcacc attgagttta aacccgctga tcagcctcga ctgtgccttc tagttgccag
     4741 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     4801 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
     4861 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat
     4921 gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctcgata
     4981 ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     5041 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctag
     5101 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     5161 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     5221 ttgcgtattg ggcgcacttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     5281 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     5341 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     5401 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     5461 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     5521 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     5581 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     5641 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     5701 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     5761 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     5821 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     5881 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     5941 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     6001 tctcaagaag atcctttgat cttttctacg gggtctgaca ctcagtggaa cgaaaactca
     6061 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     6121 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     6181 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     6241 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     6301 gctgcaatga taccgcggga cccacgctca ccggctccag atttatcagc aataaaccag
     6361 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     6421 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     6481 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     6541 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     6601 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     6661 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     6721 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     6781 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     6841 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     6901 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     6961 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     7021 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     7081 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     7141 cgcacatttc cccgaaaagt gccacctgac gtcgacggat cgggagatcg atctcccgat
     7201 cccctagggt cgactctcag tacaatctgc tctgatgccg catagttaag ccagtatctg
     7261 ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta agctacaaca
     7321 aggcaaggct tgaccgacaa ttgcatgaag aatctgctta gggttaggcg ttttgcgctg
     7381 cttcgcgatg tacgggccag atatacgcgt
//
