LOCUS       sequence-396699-        7410 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of domain inlaid Nme2.Smu-ABE8e-i8.
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: pEJS2424_Nme2.Smu-ABE8e-i8
FEATURES             Location/Qualifiers
     source          1..7410
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     610..629
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        610..628
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             678..698
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      4167..4176
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4629..4649
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4658..4675
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4698..4715)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4704..4928
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4999..5015)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4999..5015)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5012..5034)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5023..5039
                     /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(5047..5053,5054..5071,5072..5077))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5092..5113
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5230..5247)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5401..5989)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5481..5500)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6160..6951,6952..7020))
                     /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     6783..6802
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7021..7125)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7204..7223)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(7395..7410,1..364)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagagatc
      601 cgcggccgct aatacgactc actataggga gagccgccac catgaaacgg acagccgacg
      661 gaagcgagtt cgagtcacca aagaagaagc ggaaagtcga agatatggcc gccttcaagc
      721 ctaacccaat caattacatc ctgggactgg ccatcggaat cgcatccgtg ggatgggcta
      781 tggtggagat cgacgaggag gagaatccta tccggctgat cgatctgggc gtgagagtgt
      841 ttgagagggc cgaggtgcca aagaccggcg attctctggc tatggcccgg agactggcac
      901 ggagcgtgag gcgcctgaca cggagaaggg cacacaggct gctgagggca cgccggctgc
      961 tgaagagaga gggcgtgctg caggcagcag acttcgatga gaatggcctg atcaagagcc
     1021 tgccaaacac cccctggcag ctgagagcag ccgccctgga caggaagctg acaccactgg
     1081 agtggtctgc cgtgctgctg cacctgatca agcaccgcgg ctacctgagc cagcggaaga
     1141 acgagggaga gacagcagac aaggagctgg gcgccctgct gaagggagtg gccaacaatg
     1201 cccacgccct gcagaccggc gatttcagga cacctgccga gctggccctg aataagtttg
     1261 agaaggagtc cggccacatc agaaaccaga ggggcgacta tagccacacc ttctcccgca
     1321 aggatctgca ggccgagctg atcctgctgt tcgagaagca gaaggagttt ggcaatccac
     1381 acgtgagcgg aggcctgaag gagggaatcg agaccctgct gatgacacag aggcctgccc
     1441 tgtccggcga cgcagtgcag aagatgctgg gacactgcac cttcgagcct gcagagccaa
     1501 aggccgccaa gaacacctac acagccgagc ggtttatctg gctgacaaag ctgaacaatc
     1561 tgagaatcct ggagcaggga tccgagaggc cactgaccga cacagagagg gccaccctga
     1621 tggatgagcc ttaccggaag tctaagctga catatgccca ggccagaaag ctgctgggcc
     1681 tggaggacac cgccttcttt aagggcctga gatacggcaa ggataatgcc gaggcctcca
     1741 cactgatgga gatgaaggcc tatcacgcca tctctcgcgc cctggagaag gagggcctga
     1801 aggacaagaa gtcccccctg aacctgagct ccgagctgca ggatgagatc ggcaccgcct
     1861 tctctctgtt taagaccgac gaggatatca caggccgcct gaaggacagg gtgcagcctg
     1921 agatcctgga ggccctgctg aagcacatct ctttcgataa gtttgtgcag atcagcctga
     1981 aggccctgag aaggatcgtg ccactgatgg agcagggcaa gcggtacgac gaggcctgcg
     2041 ccgagatcta cggcgatcac tatggcaaga agaacacaga ggagaagatc tatctgcccc
     2101 ctatccctgc cgacgagatc agaaatcctg tggtgctgag ggccctgtcc caggcaagaa
     2161 aagtgatcaa cggagtggtg cgccggtacg gatctccagc ccggatccac atcgagaccg
     2221 ccagagaagt gggcaagagc ttcaaggacc ggaaggagat cgagaagaga caggaggaga
     2281 atcgcaagga tcgggagaag gccgccgcca agtttaggga gtacttccct aactttgtgg
     2341 gcgagccaaa gtctaaggac atcctgaagc tgcgcctgta cgagcagcag cacggcaagt
     2401 gtctgtatag cggcaaggag atcaatctgg tgcggctgaa cgagaagggc tatgtggaga
     2461 tcgatcacgc cctgcctttc tccagaacct gggacgattc ttttaacaat aaggtgctgg
     2521 tgctgggcag cgagaaccag aataagggca atcagacacc atacgagtat ttcaatggca
     2581 aggacaactc cagggagtgg caggagttca aggcccgcgt ggagacctct agatttccca
     2641 ggagcaagaa gcagcggatc ctgctgcaga agttcgacga ggatggcttt aaggagtgca
     2701 acctgaatga caccagatac gtgaaccggt tcctgtgcca gtttgtggcc gatcacatcc
     2761 tgctgaccgg caagggcaag agaagggtgt tcgcctctaa tggccagatc acaaacctgc
     2821 tgaggggatt ttggggactg aggaaggtgc gggcagagaa tgacagacac cacgcactgg
     2881 atgcagtggt ggtggcatgc agcaccgtgg caatgcagca gaagatcaca agattcgtga
     2941 ggtataagga gatgaacgcc tttgacggca agaccatcga taaggagaca ggcaaggtgc
     3001 tgcaccagaa gacccacttc ccccagcctt gggagttctt tgcccaggaa gtgatgatcc
     3061 gggtgttcgg caagccagac ggcaagcctg gcggatcagg aggctctggc ggttcaggtg
     3121 gatcaggcgg tagcggaggt tcaggtggtt ctgaggtgga gttttcccac gagtactgga
     3181 tgagacatgc cctgaccctg gccaagaggg cacgcgatga gagggaggtg cctgtgggag
     3241 ccgtgctggt gctgaacaat agagtgatcg gcgagggctg gaacagagcc atcggcctgc
     3301 acgacccaac agcccatgcc gaaattatgg ccctgagaca gggcggcctg gtcatgcaga
     3361 actacagact gattgacgcc accctgtacg tgacattcga gccttgcgtg atgtgcgccg
     3421 gcgccatgat ccactctagg atcggccgcg tggtgtttgg cgtgaggaac agcaaacggg
     3481 gcgccgcagg ctccctgatg aacgtgctga actaccccgg catgaatcac cgcgtcgaaa
     3541 ttaccgaggg aatcctggca gatgaatgtg ccgccctgct gtgcgacttc taccggatgc
     3601 ctagacaggt gttcaatgct cagaagaagg cccagagctc catcaacggc tcctctggct
     3661 ctgagacacc tggcacaagc gagagcgcaa cacctgaaag cagcggcgag tttgaggagg
     3721 ccgatacccc agagaagctg aggacactgc tggcagagaa gctgtctagc aggccagagg
     3781 cagtgcacga gtacgtgacc ccactgttcg tgtccagggc acccaatcgg aagatgtctg
     3841 gcgcccacaa ggacacactg agaagcgcca agaggtttgt gaagcacaac gagaagatct
     3901 ccgtgaagag agtgtggctg accgagatca agctggccga tctggagaac atggtgaatt
     3961 acaagaacgg cagggagatc gagctgtatg aggccctgaa ggcaaggctg gaggcctacg
     4021 gaggaaatgc caagcaggcc ttcgacccaa aggataaccc cttttataag aagggaggac
     4081 agctggtgaa ggccgtgcgg gtggagaaga cccaggagag cggcgtgctg ctgaataaga
     4141 agaacgccta cacaatcgcc gacaacgcca ccatggtgcg ggtggacgtg tacaccaagg
     4201 ccggcaagaa ctacctggtt cctgtgtacg tgtggcaggt ggcccagggc atcttaccca
     4261 accgcgccgt gaccagcggc aagtccgagg ctgactggga cctgatcgat gagagcttcg
     4321 agttcaagtt ctctctgtcc cggggagatc tcgtggaaat gatctccaac aagggcagaa
     4381 tcttcggcta ctacaacggc ctggacagag ccaacggctc tattggaatt agagagcacg
     4441 acctagagaa gagcaagggc aaagacggcg tgcatagagt gggagtgaaa acagctacag
     4501 catttaacaa gtaccacgtg gatcccctgg gcaaagagat ccacagatgc agcagcgaac
     4561 ccagacctac actgaaaatc aagtctaaga aggaggataa aagaaccgcc gacggcagcg
     4621 aattcgagcc caagaagaag aggaaagtct aaccggtcat catcaccatc accattgagt
     4681 ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc
     4741 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa
     4801 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg
     4861 gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg
     4921 ctctatggct tctgaggcgg aaagaaccag ctggggctcg ataccgtcga cctctagcta
     4981 gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     5041 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tagggtgcct aatgagtgag
     5101 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     5161 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcac
     5221 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     5281 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     5341 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     5401 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     5461 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     5521 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     5581 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     5641 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     5701 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     5761 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     5821 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     5881 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     5941 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     6001 gatcttttct acggggtctg acactcagtg gaacgaaaac tcacgttaag ggattttggt
     6061 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     6121 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     6181 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     6241 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     6301 ggacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     6361 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     6421 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     6481 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     6541 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     6601 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     6661 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     6721 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     6781 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     6841 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     6901 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     6961 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     7021 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     7081 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     7141 agtgccacct gacgtcgacg gatcgggaga tcgatctccc gatcccctag ggtcgactct
     7201 cagtacaatc tgctctgatg ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt
     7261 ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag gcttgaccga
     7321 caattgcatg aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc
     7381 cagatatacg cgttgacatt gattattgac
//
