LOCUS       sequence-394724-        7467 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  All-in-one AAV plasmid expressing Nme2-ABE8e-i1 and U6 sgRNA 
            cassette. SapI enables cloning new spacers..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7467)
  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 7467)
  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: pEJS2601_AAV-Nme2-ABE8e-i1-U6-2xSapI
FEATURES             Location/Qualifiers
     source          1..7467
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     CDS             447..467
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4374..4394
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     promoter        4458..4706
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     4458..4478
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4629..4648
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     repeat_region   4843..4983
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4843..4972
                     /label=AAV2 ITR
     rep_origin      5058..5513
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5145..5164)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5355..5376
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5530..5549)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5682..5786
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5787..5855,5856..6647)
                     /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     complement(6005..6024)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6818..7406
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7307..7326
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggcctcta gagtttatgg aggcggtact atgtagatga gaattcagga
      181 gcaaactggg aaaagcaact gcttccaaat atttgtgatt tttacagtgt agttttggaa
      241 aaactcttag cctaccaatt cttctaagtg ttttaaaatg tgggagccag tacacatgaa
      301 gttatagagt gttttaatga ggcttaaata tttaccgtaa ctatgaaatg ctacgcatat
      361 catgctgttc aggctccgtg gccacgcaac tcatactaag agccgccacc atgaaacgga
      421 cagccgacgg aagcgagttc gagtcaccaa agaagaagcg gaaagtcgaa gatatggccg
      481 ccttcaagcc taacccaatc aattacatcc tgggactggc catcggaatc gcatccgtgg
      541 gatgggctat ggtggagatc gacgaggagg agaatcctat ccggctgatc gatctgggcg
      601 tgagagtgtt tgagagggcc gaggtgccaa agaccggcga ttctctggct atggcccgga
      661 gactggcacg gagcgtgagg cgcctgacac ggagaagggc acacaggctg ctgagggcac
      721 gccggctgct gaagagagag ggcgtgctgc aggcagcaga cttcgatgag aatggcctga
      781 tcaagagcct gccaaacacc ccctggcagc tgagagcagc cgccctggac aggaagctga
      841 caccactgga gtggtctgcc gtgctgctgc acctgatcaa gcaccgcggc tacctgagcc
      901 agcggaagaa cgagggagag acagcagaca aggagctggg cgccctgctg aagggagtgg
      961 ccaacaatgc ccacgccctg cagaccggcg atttcaggac acctgccgag ctggccctga
     1021 ataagtttga gaaggagtcc ggccacatca gaaaccagag gggcgactat agccacacct
     1081 tctcccgcaa ggatctgcag gccgagctga tcctgctgtt cgagaagcag aaggagtttg
     1141 gcaatccaca cgtgagcgga ggcctgaagg agggaatcga gaccctgctg atgacacaga
     1201 ggcctgccct gtccggcgac gcagtgcaga agatgctggg acactgcacc ttcgagcctg
     1261 cagagccaaa ggccgccaag aacacctaca cagccgagcg gtttatctgg ctgacaaagc
     1321 tgaacaatct gagaatcctg gagcagggcg gatcaggagg ctctggcggt tcaggtggat
     1381 caggcggtag cggaggttca ggtggttctg aggtggagtt ttcccacgag tactggatga
     1441 gacatgccct gaccctggcc aagagggcac gcgatgagag ggaggtgcct gtgggagccg
     1501 tgctggtgct gaacaataga gtgatcggcg agggctggaa cagagccatc ggcctgcacg
     1561 acccaacagc ccatgccgaa attatggccc tgagacaggg cggcctggtc atgcagaact
     1621 acagactgat tgacgccacc ctgtacgtga cattcgagcc ttgcgtgatg tgcgccggcg
     1681 ccatgatcca ctctaggatc ggccgcgtgg tgtttggcgt gaggaacagc aaacggggcg
     1741 ccgcaggctc cctgatgaac gtgctgaact accccggcat gaatcaccgc gtcgaaatta
     1801 ccgagggaat cctggcagat gaatgtgccg ccctgctgtg cgacttctac cggatgccta
     1861 gacaggtgtt caatgctcag aagaaggccc agagctccat caacggctcc tctggctctg
     1921 agacacctgg cacaagcgag agcgcaacac ctgaaagcag cggcggatcc gagaggccac
     1981 tgaccgacac agagagggcc accctgatgg atgagcctta ccggaagtct aagctgacat
     2041 atgcccaggc cagaaagctg ctgggcctgg aggacaccgc cttctttaag ggcctgagat
     2101 acggcaagga taatgccgag gcctccacac tgatggagat gaaggcctat cacgccatct
     2161 ctcgcgccct ggagaaggag ggcctgaagg acaagaagtc ccccctgaac ctgagctccg
     2221 agctgcagga tgagatcggc accgccttct ctctgtttaa gaccgacgag gatatcacag
     2281 gccgcctgaa ggacagggtg cagcctgaga tcctggaggc cctgctgaag cacatctctt
     2341 tcgataagtt tgtgcagatc agcctgaagg ccctgagaag gatcgtgcca ctgatggagc
     2401 agggcaagcg gtacgacgag gcctgcgccg agatctacgg cgatcactat ggcaagaaga
     2461 acacagagga gaagatctat ctgcccccta tccctgccga cgagatcaga aatcctgtgg
     2521 tgctgagggc cctgtcccag gcaagaaaag tgatcaacgg agtggtgcgc cggtacggat
     2581 ctccagcccg gatccacatc gagaccgcca gagaagtggg caagagcttc aaggaccgga
     2641 aggagatcga gaagagacag gaggagaatc gcaaggatcg ggagaaggcc gccgccaagt
     2701 ttagggagta cttccctaac tttgtgggcg agccaaagtc taaggacatc ctgaagctgc
     2761 gcctgtacga gcagcagcac ggcaagtgtc tgtatagcgg caaggagatc aatctggtgc
     2821 ggctgaacga gaagggctat gtggagatcg atcacgccct gcctttctcc agaacctggg
     2881 acgattcttt taacaataag gtgctggtgc tgggcagcga gaaccagaat aagggcaatc
     2941 agacaccata cgagtatttc aatggcaagg acaactccag ggagtggcag gagttcaagg
     3001 cccgcgtgga gacctctaga tttcccagga gcaagaagca gcggatcctg ctgcagaagt
     3061 tcgacgagga tggctttaag gagtgcaacc tgaatgacac cagatacgtg aaccggttcc
     3121 tgtgccagtt tgtggccgat cacatcctgc tgaccggcaa gggcaagaga agggtgttcg
     3181 cctctaatgg ccagatcaca aacctgctga ggggattttg gggactgagg aaggtgcggg
     3241 cagagaatga cagacaccac gcactggatg cagtggtggt ggcatgcagc accgtggcaa
     3301 tgcagcagaa gatcacaaga ttcgtgaggt ataaggagat gaacgccttt gacggcaaga
     3361 ccatcgataa ggagacaggc aaggtgctgc accagaagac ccacttcccc cagccttggg
     3421 agttctttgc ccaggaagtg atgatccggg tgttcggcaa gccagacggc aagcctgagt
     3481 ttgaggaggc cgatacccca gagaagctga ggacactgct ggcagagaag ctgtctagca
     3541 ggccagaggc agtgcacgag tacgtgaccc cactgttcgt gtccagggca cccaatcgga
     3601 agatgtctgg cgcccacaag gacacactga gaagcgccaa gaggtttgtg aagcacaacg
     3661 agaagatctc cgtgaagaga gtgtggctga ccgagatcaa gctggccgat ctggagaaca
     3721 tggtgaatta caagaacggc agggagatcg agctgtatga ggccctgaag gcaaggctgg
     3781 aggcctacgg aggaaatgcc aagcaggcct tcgacccaaa ggataacccc ttttataaga
     3841 agggaggaca gctggtgaag gccgtgcggg tggagaagac ccaggagagc ggcgtgctgc
     3901 tgaataagaa gaacgcctac acaatcgccg acaatggcga tatggtgaga gtggacgtgt
     3961 tctgtaaggt ggataagaag ggcaagaatc agtactttat cgtgcctatc tatgcctggc
     4021 aggtggccga gaacatcctg ccagacatcg attgcaaggg ctacagaatc gacgatagct
     4081 atacattctg tttttccctg cacaagtatg acctgatcgc cttccagaag gatgagaagt
     4141 ccaaggtgga gtttgcctac tatatcaatt gcgactcctc taacggcagg ttctacctgg
     4201 cctggcacga taagggcagc aaggagcagc agtttcgcat ctccacccag aatctggtgc
     4261 tgatccagaa gtatcaggtg aacgagctgg gcaaggagat caggccatgt cggctgaaga
     4321 agcgcccacc cgtgcgggag gataaaagaa ccgccgacgg cagcgaattc gagcccaaga
     4381 agaagaggaa agtctaaaat aaaagatctt tattttcatt agatctgtgt gttggttttt
     4441 tgtgtaagcg ctctagagag ggcctatttc ccatgattcc ttcatatttg catatacgat
     4501 acaaggctgt tagagagata attagaatta atttgactgt aaacacaaag atattagtac
     4561 aaaatacgtg acgtagaaag taataatttc ttgggtagtt tgcagtttta aaattatgtt
     4621 ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta tttcgatttc ttggctttat
     4681 atatcttgtg gaaaggacga aacaccggaa gagcattcca gctcttcagt tgtagctccc
     4741 gaaacgttgc tacaataagg ccgtctgaaa agatgtgccg caacgctctg ccccttaaag
     4801 cttctgcttt aaggggcatc gtttattttt ttgtttaaac gcaggaaccc ctagtgatgg
     4861 agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg
     4921 cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc
     4981 aggggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatac
     5041 gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     5101 tacgcgcagc gtgaccgcta cacttgccag cgccttagcg cccgctcctt tcgctttctt
     5161 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     5221 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg atttgggtga
     5281 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     5341 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaact ctatctcggg
     5401 ctattctttt gatttataag ggattttgcc gatttcggtc tattggttaa aaaatgagct
     5461 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa ttttatggtg
     5521 cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac
     5581 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     5641 gctcgagtta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     5701 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     5761 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     5821 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     5881 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     5941 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     6001 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
     6061 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     6121 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     6181 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     6241 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     6301 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
     6361 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     6421 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     6481 aatctggagc cggtgagcgt ggaagccgcg gtatcattgc agcactgggg ccagatggta
     6541 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     6601 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     6661 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     6721 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     6781 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     6841 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     6901 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     6961 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     7021 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     7081 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     7141 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     7201 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     7261 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     7321 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     7381 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     7441 ccttttgctg gccttttgct cacatgt
//
