LOCUS       sequence-394672-        7469 bp    DNA     circular SYN 14-APR-2025
DEFINITION  All-in-one AAV plasmid expressing Nme2-ABE8e-i1 and U6 driven sgRNA 
            targeting the mouse Rosa26 gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7469)
  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 7469)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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: pEJS2603_AAV-Nme2-ABE8e-i1-U6-Rosa26
FEATURES             Location/Qualifiers
     source          1..7469
                     /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   4845..4985
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4845..4974
                     /label=AAV2 ITR
     rep_origin      5060..5515
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5147..5166)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5357..5378
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5532..5551)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5684..5788
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5789..5857,5858..6649)
                     /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(6007..6026)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6820..7408
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7309..7328
                     /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 aacaccggag gaccgccctg ggcctgggag gttgtagctc
     4741 ccgaaacgtt gctacaataa ggccgtctga aaagatgtgc cgcaacgctc tgccccttaa
     4801 agcttctgct ttaaggggca tcgtttattt ttttgtttaa acgcaggaac ccctagtgat
     4861 ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt
     4921 cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct
     4981 gcaggggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat
     5041 acgtcaaagc aaccatagta cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     5101 gttacgcgca gcgtgaccgc tacacttgcc agcgccttag cgcccgctcc tttcgctttc
     5161 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     5221 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgatttgggt
     5281 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     5341 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ctctatctcg
     5401 ggctattctt ttgatttata agggattttg ccgatttcgg tctattggtt aaaaaatgag
     5461 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac aattttatgg
     5521 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca
     5581 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     5641 gtgctcgagt tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     5701 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     5761 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     5821 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     5881 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     5941 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     6001 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     6061 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     6121 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     6181 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     6241 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     6301 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
     6361 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     6421 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     6481 taaatctgga gccggtgagc gtggaagccg cggtatcatt gcagcactgg ggccagatgg
     6541 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     6601 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     6661 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     6721 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     6781 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     6841 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     6901 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     6961 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     7021 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     7081 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     7141 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     7201 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     7261 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     7321 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     7381 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     7441 ggccttttgc tggccttttg ctcacatgt
//
