LOCUS       sequence-394774-        7854 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of domain inlaid Nme2-rAPOBEC-i8.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7854)
  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 7854)
  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: pEJS2216_Nme2-rAPOBEC-i8
FEATURES             Location/Qualifiers
     source          1..7854
                     /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             3147..3830
                     /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             4782..5030
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             5073..5093
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5102..5119
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5142..5159)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5148..5372
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5443..5459)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5443..5459)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5456..5478)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5467..5483
                     /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(5491..5497,5498..5515,5516..5521))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5536..5557
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5674..5691)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5845..6433)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5925..5944)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6604..7395,7396..7464))
                     /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     7227..7246
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7465..7569)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7648..7667)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(7839..7854,1..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 ggtggtagct cagagactgg cccagtggct gtggacccca
     3181 cattgagacg gcggatcgag ccccatgagt ttgaggtatt cttcgatccg agagagctcc
     3241 gcaaggagac ctgcctgctt tacgaaatta attggggggg ccggcactcc atttggcgac
     3301 atacatcaca gaacactaac aagcacgtcg aagtcaactt catcgagaag ttcacgacag
     3361 aaagatattt ctgtccgaac acaaggtgca gcattacctg gtttctcagc tggagcccat
     3421 gcggcgaatg tagtagggcc atcactgaat tcctgtcaag gtatccccac gtcactctgt
     3481 ttatttacat cgcaaggctg taccaccacg ctgacccccg caatcgacaa ggcctgcggg
     3541 atttgatctc ttcaggtgtg actatccaaa ttatgactga gcaggagtca ggatactgct
     3601 ggagaaactt tgtgaattat agcccgagta atgaagccca ctggcctagg tatccccatc
     3661 tgtgggtacg actgtacgtt cttgaactgt actgcatcat actgggcctg cctccttgtc
     3721 tcaacattct gagaaggaag cagccacagc tgacattctt taccatcgct cttcagtctt
     3781 gtcattacca gcgactgccc ccacacattc tctgggccac cgggttgaaa ggctcctctg
     3841 gctctgagac acctggcaca agcgagagcg caacacctga aagcagcggc gagtttgagg
     3901 aggccgatac cccagagaag ctgaggacac tgctggcaga gaagctgtct agcaggccag
     3961 aggcagtgca cgagtacgtg accccactgt tcgtgtccag ggcacccaat cggaagatgt
     4021 ctggcgccca caaggacaca ctgagaagcg ccaagaggtt tgtgaagcac aacgagaaga
     4081 tctccgtgaa gagagtgtgg ctgaccgaga tcaagctggc cgatctggag aacatggtga
     4141 attacaagaa cggcagggag atcgagctgt atgaggccct gaaggcaagg ctggaggcct
     4201 acggaggaaa tgccaagcag gccttcgacc caaaggataa ccccttttat aagaagggag
     4261 gacagctggt gaaggccgtg cgggtggaga agacccagga gagcggcgtg ctgctgaata
     4321 agaagaacgc ctacacaatc gccgacaatg gcgatatggt gagagtggac gtgttctgta
     4381 aggtggataa gaagggcaag aatcagtact ttatcgtgcc tatctatgcc tggcaggtgg
     4441 ccgagaacat cctgccagac atcgattgca agggctacag aatcgacgat agctatacat
     4501 tctgtttttc cctgcacaag tatgacctga tcgccttcca gaaggatgag aagtccaagg
     4561 tggagtttgc ctactatatc aattgcgact cctctaacgg caggttctac ctggcctggc
     4621 acgataaggg cagcaaggag cagcagtttc gcatctccac ccagaatctg gtgctgatcc
     4681 agaagtatca ggtgaacgag ctgggcaagg agatcaggcc atgtcggctg aagaagcgcc
     4741 cacccgtgcg gagcggagga tccggaggat ctggaggcag caccaacctg tctgacatca
     4801 tcgagaagga gacaggcaag cagctggtca tccaggagag catcctgatg ctgcccgaag
     4861 aagtcgaaga agtgatcgga aacaagcctg agagcgatat cctggtccat accgcctacg
     4921 acgagagtac cgacgaaaat gtgatgctgc tgacatccga cgccccagag tataagccct
     4981 gggctctggt catccaggat tccaacggag agaacaaaat caaaatgctg tctggcggct
     5041 caaaaagaac cgccgacggc agcgaattcg agcccaagaa gaagaggaaa gtctaaccgg
     5101 tcatcatcac catcaccatt gagtttaaac ccgctgatca gcctcgactg tgccttctag
     5161 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac
     5221 tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca
     5281 ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag
     5341 caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg
     5401 ctcgataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt
     5461 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     5521 agcctagggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     5581 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     5641 aggcggtttg cgtattgggc gcacttccgc ttcctcgctc actgactcgc tgcgctcggt
     5701 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     5761 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     5821 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     5881 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     5941 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     6001 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     6061 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     6121 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     6181 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     6241 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     6301 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     6361 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     6421 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacactc agtggaacga
     6481 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     6541 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     6601 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     6661 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     6721 ccccagtgct gcaatgatac cgcgggaccc acgctcaccg gctccagatt tatcagcaat
     6781 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     6841 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     6901 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     6961 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     7021 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     7081 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     7141 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     7201 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     7261 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     7321 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     7381 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcagaaaagg gaataagggc
     7441 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     7501 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     7561 ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc gacggatcgg gagatcgatc
     7621 tcccgatccc ctagggtcga ctctcagtac aatctgctct gatgccgcat agttaagcca
     7681 gtatctgctc cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca aaatttaagc
     7741 tacaacaagg caaggcttga ccgacaattg catgaagaat ctgcttaggg ttaggcgttt
     7801 tgcgctgctt cgcgatgtac gggccagata tacgcgttga cattgattat tgac
//
