LOCUS       sequence-394753-        7653 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of domain inlaid Nme2-evoFERNY-i8.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7653)
  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 7653)
  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: pEJS2197_Nme2-evoFERNY-i8
FEATURES             Location/Qualifiers
     source          1..7653
                     /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"
     CDS             4581..4829
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             4872..4892
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4901..4918
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4941..4958)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4947..5171
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5242..5258)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5242..5258)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5255..5277)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5266..5282
                     /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(5290..5296,5297..5314,5315..5320))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5335..5356
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5473..5490)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5644..6232)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5724..5743)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6403..7194,7195..7263))
                     /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     7026..7045
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7264..7368)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7447..7466)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(7638..7653,1..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 agatgccgcc ttcaagccta
      721 acccaatcaa ttacatcctg ggactggcca tcggaatcgc atccgtggga tgggctatgg
      781 tggagatcga cgaggaggag aatcctatcc ggctgatcga tctgggcgtg agagtgtttg
      841 agagggccga ggtgccaaag accggcgatt ctctggctat ggcccggaga ctggcacgga
      901 gcgtgaggcg cctgacacgg agaagggcac acaggctgct gagggcacgc cggctgctga
      961 agagagaggg cgtgctgcag gcagcagact tcgatgagaa tggcctgatc aagagcctgc
     1021 caaacacccc ctggcagctg agagcagccg ccctggacag gaagctgaca ccactggagt
     1081 ggtctgccgt gctgctgcac ctgatcaagc accgcggcta cctgagccag cggaagaacg
     1141 agggagagac agcagacaag gagctgggcg ccctgctgaa gggagtggcc aacaatgccc
     1201 acgccctgca gaccggcgat ttcaggacac ctgccgagct ggccctgaat aagtttgaga
     1261 aggagtccgg ccacatcaga aaccagaggg gcgactatag ccacaccttc tcccgcaagg
     1321 atctgcaggc cgagctgatc ctgctgttcg agaagcagaa ggagtttggc aatccacacg
     1381 tgagcggagg cctgaaggag ggaatcgaga ccctgctgat gacacagagg cctgccctgt
     1441 ccggcgacgc agtgcagaag atgctgggac actgcacctt cgagcctgca gagccaaagg
     1501 ccgccaagaa cacctacaca gccgagcggt ttatctggct gacaaagctg aacaatctga
     1561 gaatcctgga gcagggatcc gagaggccac tgaccgacac agagagggcc accctgatgg
     1621 atgagcctta ccggaagtct aagctgacat atgcccaggc cagaaagctg ctgggcctgg
     1681 aggacaccgc cttctttaag ggcctgagat acggcaagga taatgccgag gcctccacac
     1741 tgatggagat gaaggcctat cacgccatct ctcgcgccct ggagaaggag ggcctgaagg
     1801 acaagaagtc ccccctgaac ctgagctccg agctgcagga tgagatcggc accgccttct
     1861 ctctgtttaa gaccgacgag gatatcacag gccgcctgaa ggacagggtg cagcctgaga
     1921 tcctggaggc cctgctgaag cacatctctt tcgataagtt tgtgcagatc agcctgaagg
     1981 ccctgagaag gatcgtgcca ctgatggagc agggcaagcg gtacgacgag gcctgcgccg
     2041 agatctacgg cgatcactat ggcaagaaga acacagagga gaagatctat ctgcccccta
     2101 tccctgccga cgagatcaga aatcctgtgg tgctgagggc cctgtcccag gcaagaaaag
     2161 tgatcaacgg agtggtgcgc cggtacggat ctccagcccg gatccacatc gagaccgcca
     2221 gagaagtggg caagagcttc aaggaccgga aggagatcga gaagagacag gaggagaatc
     2281 gcaaggatcg ggagaaggcc gccgccaagt ttagggagta cttccctaac tttgtgggcg
     2341 agccaaagtc taaggacatc ctgaagctgc gcctgtacga gcagcagcac ggcaagtgtc
     2401 tgtatagcgg caaggagatc aatctggtgc ggctgaacga gaagggctat gtggagatcg
     2461 atcacgccct gcctttctcc agaacctggg acgattcttt taacaataag gtgctggtgc
     2521 tgggcagcga gaaccagaat aagggcaatc agacaccata cgagtatttc aatggcaagg
     2581 acaactccag ggagtggcag gagttcaagg cccgcgtgga gacctctaga tttcccagga
     2641 gcaagaagca gcggatcctg ctgcagaagt tcgacgagga tggctttaag gagtgcaacc
     2701 tgaatgacac cagatacgtg aaccggttcc tgtgccagtt tgtggccgat cacatcctgc
     2761 tgaccggcaa gggcaagaga agggtgttcg cctctaatgg ccagatcaca aacctgctga
     2821 ggggattttg gggactgagg aaggtgcggg cagagaatga cagacaccac gcactggatg
     2881 cagtggtggt ggcatgcagc accgtggcaa tgcagcagaa gatcacaaga ttcgtgaggt
     2941 ataaggagat gaacgccttt gacggcaaga ccatcgataa ggagacaggc aaggtgctgc
     3001 accagaagac ccacttcccc cagccttggg agttctttgc ccaggaagtg atgatccggg
     3061 tgttcggcaa gccagacggc aagcctggcg gatcaggagg ctctggcggt tcaggtggat
     3121 caggcggtag cggaggttca ggtggttttg agaggaacta cgacccccgg gagctgagaa
     3181 aggagacata cctgctgtat gagatcaagt ggggcaagtc cggcaagctg tggaggcact
     3241 ggtgccagaa caatcgcaca cagcacgccg aggtgtactt cctggagaac atctttaatg
     3301 cccggagatt caatccatct acccactgta gcatcacatg gtatctgagc tggtccccct
     3361 gcgccgagtg ttctcagaag atcgtggatt tcctgaagga gcaccctaac gtgaatctgg
     3421 agatctatgt ggcccggctg tactatccag agaacgagag gaataggcag ggcctgcggg
     3481 atctggtgaa ttccggcgtg accatcagaa tcatggacct gccagattac aactattgct
     3541 ggaagacctt cgtgagcgat cagggaggcg acgaggatta ctggccagga cacttcgccc
     3601 cttggatcaa gcagtatagc ctgaagctgg gctcctctgg ctctgagaca cctggcacaa
     3661 gcgagagcgc aacacctgaa agcagcggcg agtttgagga ggccgatacc ccagagaagc
     3721 tgaggacact gctggcagag aagctgtcta gcaggccaga ggcagtgcac gagtacgtga
     3781 ccccactgtt cgtgtccagg gcacccaatc ggaagatgtc tggcgcccac aaggacacac
     3841 tgagaagcgc caagaggttt gtgaagcaca acgagaagat ctccgtgaag agagtgtggc
     3901 tgaccgagat caagctggcc gatctggaga acatggtgaa ttacaagaac ggcagggaga
     3961 tcgagctgta tgaggccctg aaggcaaggc tggaggccta cggaggaaat gccaagcagg
     4021 ccttcgaccc aaaggataac cccttttata agaagggagg acagctggtg aaggccgtgc
     4081 gggtggagaa gacccaggag agcggcgtgc tgctgaataa gaagaacgcc tacacaatcg
     4141 ccgacaatgg cgatatggtg agagtggacg tgttctgtaa ggtggataag aagggcaaga
     4201 atcagtactt tatcgtgcct atctatgcct ggcaggtggc cgagaacatc ctgccagaca
     4261 tcgattgcaa gggctacaga atcgacgata gctatacatt ctgtttttcc ctgcacaagt
     4321 atgacctgat cgccttccag aaggatgaga agtccaaggt ggagtttgcc tactatatca
     4381 attgcgactc ctctaacggc aggttctacc tggcctggca cgataagggc agcaaggagc
     4441 agcagtttcg catctccacc cagaatctgg tgctgatcca gaagtatcag gtgaacgagc
     4501 tgggcaagga gatcaggcca tgtcggctga agaagcgccc acccgtgcgg agcggaggat
     4561 ccggaggatc tggaggcagc accaacctgt ctgacatcat cgagaaggag acaggcaagc
     4621 agctggtcat ccaggagagc atcctgatgc tgcccgaaga agtcgaagaa gtgatcggaa
     4681 acaagcctga gagcgatatc ctggtccata ccgcctacga cgagagtacc gacgaaaatg
     4741 tgatgctgct gacatccgac gccccagagt ataagccctg ggctctggtc atccaggatt
     4801 ccaacggaga gaacaaaatc aaaatgctgt ctggcggctc aaaaagaacc gccgacggca
     4861 gcgaattcga gcccaagaag aagaggaaag tctaaccggt catcatcacc atcaccattg
     4921 agtttaaacc cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg
     4981 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata
     5041 aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt
     5101 ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt
     5161 gggctctatg gcttctgagg cggaaagaac cagctggggc tcgataccgt cgacctctag
     5221 ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     5281 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctagggtg cctaatgagt
     5341 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     5401 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     5461 cacttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     5521 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     5581 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     5641 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     5701 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     5761 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     5821 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     5881 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     5941 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     6001 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     6061 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     6121 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     6181 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     6241 tttgatcttt tctacggggt ctgacactca gtggaacgaa aactcacgtt aagggatttt
     6301 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     6361 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     6421 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     6481 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     6541 gcgggaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     6601 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     6661 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     6721 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     6781 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     6841 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     6901 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     6961 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     7021 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     7081 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     7141 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     7201 aacaggaagg caaaatgccg cagaaaaggg aataagggcg acacggaaat gttgaatact
     7261 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     7321 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     7381 aaaagtgcca cctgacgtcg acggatcggg agatcgatct cccgatcccc tagggtcgac
     7441 tctcagtaca atctgctctg atgccgcata gttaagccag tatctgctcc ctgcttgtgt
     7501 gttggaggtc gctgagtagt gcgcgagcaa aatttaagct acaacaaggc aaggcttgac
     7561 cgacaattgc atgaagaatc tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg
     7621 ggccagatat acgcgttgac attgattatt gac
//
