LOCUS       sequence-389819-        7471 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Optimized single AAV vector for expressing N-terminal fusion 
            Nme2Cas9-ABE8e and one U6 driven sgRNA targeting mouse Rosa26 gene. 
            This optimized construct showed improved in vivo editing 
            efficiency..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7471)
  AUTHORS   Zhang H, Bamidele N, Liu P, Ojelabi O, Gao XD, Rodriguez T, Cheng H,
            Kelly K, Watts JK, Xie J, Gao G, Wolfe SA, Xue W, Sontheimer EJ
  TITLE     Adenine Base Editing In Vivo with a Single Adeno-Associated Virus 
            Vector.
  JOURNAL   GEN Biotechnol. 2022 Jun 1;1(3):285-299. doi: 
            10.1089/genbio.2022.0015. Epub 2022 Jun 14.
  PUBMED    35811581
REFERENCE   2  (bases 1 to 7471)
  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"; doi: 
            "10.1089/genbio.2022.0015"; journalName: "GEN Biotechnol"; date: 
            "2022-06-1- 1"; volume: "1"; issue: "3"; pages: "285-299"
COMMENT     Sequence Label: pEJS-HZ79 All-in-one
            AAV-U1a-NmeABE8e-2xBPSV40...
FEATURES             Location/Qualifiers
     source          1..7471
                     /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             443..463
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4376..4396
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     promoter        4460..4708
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     4460..4480
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4631..4650
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     repeat_region   4847..4987
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4847..4976
                     /label=AAV2 ITR
     rep_origin      5062..5517
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5149..5168)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5359..5380
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5534..5553)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5686..5790
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5791..5859,5860..6651)
                     /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(6009..6028)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6822..7410
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7311..7330
                     /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 tcatactacc ggtgccacca tgaaaagaac
      421 cgccgacggc agcgaattcg agcccaagaa gaagaggaaa gtcggcggta gcggcggagg
      481 cagcggtggc ggcagcggct ctgaggtgga gttttcccac gagtactgga tgagacatgc
      541 cctgaccctg gccaagaggg cacgcgatga gagggaggtg cctgtgggag ccgtgctggt
      601 gctgaacaat agagtgatcg gcgagggctg gaacagagcc atcggcctgc acgacccaac
      661 agcccatgcc gaaattatgg ccctgagaca gggcggcctg gtcatgcaga actacagact
      721 gattgacgcc accctgtacg tgacattcga gccttgcgtg atgtgcgccg gcgccatgat
      781 ccactctagg atcggccgcg tggtgtttgg cgtgaggaac agcaaacggg gcgccgcagg
      841 ctccctgatg aacgtgctga actaccccgg catgaatcac cgcgtcgaaa ttaccgaggg
      901 aatcctggca gatgaatgtg ccgccctgct gtgcgacttc taccggatgc ctagacaggt
      961 gttcaatgct cagaagaagg cccagagctc catcaactcc ggaggatcta gcggaggctc
     1021 ctctggctct gagacacctg gcacaagcga gagcgcaaca cctgaaagca gcgggggcag
     1081 cagcgggggg tcaatggccg ccttcaagcc taacccaatc aattacatcc tgggactggc
     1141 catcggaatc gcatccgtgg gatgggctat ggtggagatc gacgaggagg agaatcctat
     1201 ccggctgatc gatctgggcg tgagagtgtt tgagagggcc gaggtgccaa agaccggcga
     1261 ttctctggct atggcccgga gactggcacg gagcgtgagg cgcctgacac ggagaagggc
     1321 acacaggctg ctgagggcac gccggctgct gaagagagag ggcgtgctgc aggcagcaga
     1381 cttcgatgag aatggcctga tcaagagcct gccaaacacc ccctggcagc tgagagcagc
     1441 cgccctggac aggaagctga caccactgga gtggtctgcc gtgctgctgc acctgatcaa
     1501 gcaccgcggc tacctgagcc agcggaagaa cgagggagag acagcagaca aggagctggg
     1561 cgccctgctg aagggagtgg ccaacaatgc ccacgccctg cagaccggcg atttcaggac
     1621 acctgccgag ctggccctga ataagtttga gaaggagtcc ggccacatca gaaaccagag
     1681 gggcgactat agccacacct tctcccgcaa ggatctgcag gccgagctga tcctgctgtt
     1741 cgagaagcag aaggagtttg gcaatccaca cgtgagcgga ggcctgaagg agggaatcga
     1801 gaccctgctg atgacacaga ggcctgccct gtccggcgac gcagtgcaga agatgctggg
     1861 acactgcacc ttcgagcctg cagagccaaa ggccgccaag aacacctaca cagccgagcg
     1921 gtttatctgg ctgacaaagc tgaacaatct gagaatcctg gagcagggat ccgagaggcc
     1981 actgaccgac acagagaggg ccaccctgat ggatgagcct taccggaagt ctaagctgac
     2041 atatgcccag gccagaaagc tgctgggcct ggaggacacc gccttcttta agggcctgag
     2101 atacggcaag gataatgccg aggcctccac actgatggag atgaaggcct atcacgccat
     2161 ctctcgcgcc ctggagaagg agggcctgaa ggacaagaag tcccccctga acctgagctc
     2221 cgagctgcag gatgagatcg gcaccgcctt ctctctgttt aagaccgacg aggatatcac
     2281 aggccgcctg aaggacaggg tgcagcctga gatcctggag gccctgctga agcacatctc
     2341 tttcgataag tttgtgcaga tcagcctgaa ggccctgaga aggatcgtgc cactgatgga
     2401 gcagggcaag cggtacgacg aggcctgcgc cgagatctac ggcgatcact atggcaagaa
     2461 gaacacagag gagaagatct atctgccccc tatccctgcc gacgagatca gaaatcctgt
     2521 ggtgctgagg gccctgtccc aggcaagaaa agtgatcaac ggagtggtgc gccggtacgg
     2581 atctccagcc cggatccaca tcgagaccgc cagagaagtg ggcaagagct tcaaggaccg
     2641 gaaggagatc gagaagagac aggaggagaa tcgcaaggat cgggagaagg ccgccgccaa
     2701 gtttagggag tacttcccta actttgtggg cgagccaaag tctaaggaca tcctgaagct
     2761 gcgcctgtac gagcagcagc acggcaagtg tctgtatagc ggcaaggaga tcaatctggt
     2821 gcggctgaac gagaagggct atgtggagat cgatcacgcc ctgcctttct ccagaacctg
     2881 ggacgattct tttaacaata aggtgctggt gctgggcagc gagaaccaga ataagggcaa
     2941 tcagacacca tacgagtatt tcaatggcaa ggacaactcc agggagtggc aggagttcaa
     3001 ggcccgcgtg gagacctcta gatttcccag gagcaagaag cagcggatcc tgctgcagaa
     3061 gttcgacgag gatggcttta aggagtgcaa cctgaatgac accagatacg tgaaccggtt
     3121 cctgtgccag tttgtggccg atcacatcct gctgaccggc aagggcaaga gaagggtgtt
     3181 cgcctctaat ggccagatca caaacctgct gaggggattt tggggactga ggaaggtgcg
     3241 ggcagagaat gacagacacc acgcactgga tgcagtggtg gtggcatgca gcaccgtggc
     3301 aatgcagcag aagatcacaa gattcgtgag gtataaggag atgaacgcct ttgacggcaa
     3361 gaccatcgat aaggagacag gcaaggtgct gcaccagaag acccacttcc cccagccttg
     3421 ggagttcttt gcccaggaag tgatgatccg ggtgttcggc aagccagacg gcaagcctga
     3481 gtttgaggag gccgataccc cagagaagct gaggacactg ctggcagaga agctgtctag
     3541 caggccagag gcagtgcacg agtacgtgac cccactgttc gtgtccaggg cacccaatcg
     3601 gaagatgtct ggcgcccaca aggacacact gagaagcgcc aagaggtttg tgaagcacaa
     3661 cgagaagatc tccgtgaaga gagtgtggct gaccgagatc aagctggccg atctggagaa
     3721 catggtgaat tacaagaacg gcagggagat cgagctgtat gaggccctga aggcaaggct
     3781 ggaggcctac ggaggaaatg ccaagcaggc cttcgaccca aaggataacc ccttttataa
     3841 gaagggagga cagctggtga aggccgtgcg ggtggagaag acccaggaga gcggcgtgct
     3901 gctgaataag aagaacgcct acacaatcgc cgacaatggc gatatggtga gagtggacgt
     3961 gttctgtaag gtggataaga agggcaagaa tcagtacttt atcgtgccta tctatgcctg
     4021 gcaggtggcc gagaacatcc tgccagacat cgattgcaag ggctacagaa tcgacgatag
     4081 ctatacattc tgtttttccc tgcacaagta tgacctgatc gccttccaga aggatgagaa
     4141 gtccaaggtg gagtttgcct actatatcaa ttgcgactcc tctaacggca ggttctacct
     4201 ggcctggcac gataagggca gcaaggagca gcagtttcgc atctccaccc agaatctggt
     4261 gctgatccag aagtatcagg tgaacgagct gggcaaggag atcaggccat gtcggctgaa
     4321 gaagcgccca cccgtgcggg aggataaaag aaccgccgac ggcagcgaat tcgagcccaa
     4381 gaagaagagg aaagtctaaa ataaaagatc tttattttca ttagatctgt gtgttggttt
     4441 tttgtgtaag cgctctagag agggcctatt tcccatgatt ccttcatatt tgcatatacg
     4501 atacaaggct gttagagaga taattagaat taatttgact gtaaacacaa agatattagt
     4561 acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg
     4621 ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt
     4681 atatatcttg tggaaaggac gaaacaccgg aggaccgccc tgggcctggg aggttgtagc
     4741 tcccgaaacg ttgctacaat aaggccgtct gaaaagatgt gccgcaacgc tctgcccctt
     4801 aaagcttctg ctttaagggg catcgtttat ttttttgttt aaacgcagga acccctagtg
     4861 atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag
     4921 gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc
     4981 ctgcaggggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     5041 atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     5101 tggttacgcg cagcgtgacc gctacacttg ccagcgcctt agcgcccgct cctttcgctt
     5161 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     5221 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgatttgg
     5281 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     5341 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aactctatct
     5401 cgggctattc ttttgattta taagggattt tgccgatttc ggtctattgg ttaaaaaatg
     5461 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaattttat
     5521 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc
     5581 caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag
     5641 ctgtgctcga gttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     5701 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     5761 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     5821 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     5881 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     5941 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     6001 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     6061 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     6121 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     6181 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     6241 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     6301 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
     6361 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     6421 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     6481 gataaatctg gagccggtga gcgtggaagc cgcggtatca ttgcagcact ggggccagat
     6541 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     6601 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     6661 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     6721 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     6781 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     6841 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     6901 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     6961 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     7021 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     7081 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     7141 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     7201 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     7261 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     7321 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     7381 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     7441 ctggcctttt gctggccttt tgctcacatg t
//
