LOCUS       sequence-389816-        7471 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Single AAV vector for expressing N-terminal fusion Nme2Cas9-ABE8e 
            and one U6 driven sgRNA targeting mouse Rosa26 gene.
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-HZ54 All-in-one
            AAV-U1a-NmeABE8e-2xBPSV40...
FEATURES             Location/Qualifiers
     source          1..7471
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(62..650)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(142..161)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(821..1612,1613..1681))
                     /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     1444..1463
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1682..1786)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1919..1938
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(1955..2410)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2092..2113)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2304..2323
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   2485..2625
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2496..2625
                     /label=AAV2 ITR
     CDS             complement(2689..2709)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(6622..6642)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     promoter        6945..7193
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     6945..6965
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     7116..7135
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     repeat_region   7342..7471
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
ORIGIN
        1 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
       61 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
      121 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
      181 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
      241 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
      301 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
      361 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
      421 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
      481 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
      541 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
      601 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
      661 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
      721 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
      781 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
      841 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
      901 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
      961 ggcttccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     1021 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     1081 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     1141 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     1201 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     1261 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     1321 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     1381 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     1441 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     1501 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     1561 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     1621 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     1681 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     1741 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     1801 aagtgccacc tgacgtctaa ctcgagcaca gcttgtctgt aagcggatgc cgggagcaga
     1861 caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggctggct taactatgcg
     1921 gcatcagagc agattgtact gagagtgcac cataaaattg taaacgttaa tattttgtta
     1981 aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaatagac cgaaatcggc
     2041 aaaatccctt ataaatcaaa agaatagccc gagatagagt tgagtgttgt tccagtttgg
     2101 aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat
     2161 cagggcgatg gcccactacg tgaaccatca cccaaatcaa gttttttggg gtcgaggtgc
     2221 cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag
     2281 ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc taaggcgctg
     2341 gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta
     2401 cagggcgcgt actatggttg ctttgacgta tgcggtgtga aataccgcac agatgcgtaa
     2461 ggagaaaata ccgcatcagg cgcccctgca ggcagctgcg cgctcgctcg ctcactgagg
     2521 ccgcccgggc aaagcccggg cgtcgggcga cctttggtcg cccggcctca gtgagcgagc
     2581 gagcgcgcag agagggagtg gccaactcca tcactagggg ttcctgcggc cgcgcttaca
     2641 caaaaaacca acacacagat ctaatgaaaa taaagatctt ttattttaga ctttcctctt
     2701 cttcttgggc tcgaattcgc tgccgtcggc ggttctttta tcctcccgca cgggtgggcg
     2761 cttcttcagc cgacatggcc tgatctcctt gcccagctcg ttcacctgat acttctggat
     2821 cagcaccaga ttctgggtgg agatgcgaaa ctgctgctcc ttgctgccct tatcgtgcca
     2881 ggccaggtag aacctgccgt tagaggagtc gcaattgata tagtaggcaa actccacctt
     2941 ggacttctca tccttctgga aggcgatcag gtcatacttg tgcagggaaa aacagaatgt
     3001 atagctatcg tcgattctgt agcccttgca atcgatgtct ggcaggatgt tctcggccac
     3061 ctgccaggca tagataggca cgataaagta ctgattcttg cccttcttat ccaccttaca
     3121 gaacacgtcc actctcacca tatcgccatt gtcggcgatt gtgtaggcgt tcttcttatt
     3181 cagcagcacg ccgctctcct gggtcttctc cacccgcacg gccttcacca gctgtcctcc
     3241 cttcttataa aaggggttat cctttgggtc gaaggcctgc ttggcatttc ctccgtaggc
     3301 ctccagcctt gccttcaggg cctcatacag ctcgatctcc ctgccgttct tgtaattcac
     3361 catgttctcc agatcggcca gcttgatctc ggtcagccac actctcttca cggagatctt
     3421 ctcgttgtgc ttcacaaacc tcttggcgct tctcagtgtg tccttgtggg cgccagacat
     3481 cttccgattg ggtgccctgg acacgaacag tggggtcacg tactcgtgca ctgcctctgg
     3541 cctgctagac agcttctctg ccagcagtgt cctcagcttc tctggggtat cggcctcctc
     3601 aaactcaggc ttgccgtctg gcttgccgaa cacccggatc atcacttcct gggcaaagaa
     3661 ctcccaaggc tgggggaagt gggtcttctg gtgcagcacc ttgcctgtct ccttatcgat
     3721 ggtcttgccg tcaaaggcgt tcatctcctt atacctcacg aatcttgtga tcttctgctg
     3781 cattgccacg gtgctgcatg ccaccaccac tgcatccagt gcgtggtgtc tgtcattctc
     3841 tgcccgcacc ttcctcagtc cccaaaatcc cctcagcagg tttgtgatct ggccattaga
     3901 ggcgaacacc cttctcttgc ccttgccggt cagcaggatg tgatcggcca caaactggca
     3961 caggaaccgg ttcacgtatc tggtgtcatt caggttgcac tccttaaagc catcctcgtc
     4021 gaacttctgc agcaggatcc gctgcttctt gctcctggga aatctagagg tctccacgcg
     4081 ggccttgaac tcctgccact ccctggagtt gtccttgcca ttgaaatact cgtatggtgt
     4141 ctgattgccc ttattctggt tctcgctgcc cagcaccagc accttattgt taaaagaatc
     4201 gtcccaggtt ctggagaaag gcagggcgtg atcgatctcc acatagccct tctcgttcag
     4261 ccgcaccaga ttgatctcct tgccgctata cagacacttg ccgtgctgct gctcgtacag
     4321 gcgcagcttc aggatgtcct tagactttgg ctcgcccaca aagttaggga agtactccct
     4381 aaacttggcg gcggccttct cccgatcctt gcgattctcc tcctgtctct tctcgatctc
     4441 cttccggtcc ttgaagctct tgcccacttc tctggcggtc tcgatgtgga tccgggctgg
     4501 agatccgtac cggcgcacca ctccgttgat cacttttctt gcctgggaca gggccctcag
     4561 caccacagga tttctgatct cgtcggcagg gatagggggc agatagatct tctcctctgt
     4621 gttcttcttg ccatagtgat cgccgtagat ctcggcgcag gcctcgtcgt accgcttgcc
     4681 ctgctccatc agtggcacga tccttctcag ggccttcagg ctgatctgca caaacttatc
     4741 gaaagagatg tgcttcagca gggcctccag gatctcaggc tgcaccctgt ccttcaggcg
     4801 gcctgtgata tcctcgtcgg tcttaaacag agagaaggcg gtgccgatct catcctgcag
     4861 ctcggagctc aggttcaggg gggacttctt gtccttcagg ccctccttct ccagggcgcg
     4921 agagatggcg tgataggcct tcatctccat cagtgtggag gcctcggcat tatccttgcc
     4981 gtatctcagg cccttaaaga aggcggtgtc ctccaggccc agcagctttc tggcctgggc
     5041 atatgtcagc ttagacttcc ggtaaggctc atccatcagg gtggccctct ctgtgtcggt
     5101 cagtggcctc tcggatccct gctccaggat tctcagattg ttcagctttg tcagccagat
     5161 aaaccgctcg gctgtgtagg tgttcttggc ggcctttggc tctgcaggct cgaaggtgca
     5221 gtgtcccagc atcttctgca ctgcgtcgcc ggacagggca ggcctctgtg tcatcagcag
     5281 ggtctcgatt ccctccttca ggcctccgct cacgtgtgga ttgccaaact ccttctgctt
     5341 ctcgaacagc aggatcagct cggcctgcag atccttgcgg gagaaggtgt ggctatagtc
     5401 gcccctctgg tttctgatgt ggccggactc cttctcaaac ttattcaggg ccagctcggc
     5461 aggtgtcctg aaatcgccgg tctgcagggc gtgggcattg ttggccactc ccttcagcag
     5521 ggcgcccagc tccttgtctg ctgtctctcc ctcgttcttc cgctggctca ggtagccgcg
     5581 gtgcttgatc aggtgcagca gcacggcaga ccactccagt ggtgtcagct tcctgtccag
     5641 ggcggctgct ctcagctgcc agggggtgtt tggcaggctc ttgatcaggc cattctcatc
     5701 gaagtctgct gcctgcagca cgccctctct cttcagcagc cggcgtgccc tcagcagcct
     5761 gtgtgccctt ctccgtgtca ggcgcctcac gctccgtgcc agtctccggg ccatagccag
     5821 agaatcgccg gtctttggca cctcggccct ctcaaacact ctcacgccca gatcgatcag
     5881 ccggatagga ttctcctcct cgtcgatctc caccatagcc catcccacgg atgcgattcc
     5941 gatggccagt cccaggatgt aattgattgg gttaggcttg aaggcggcca ttgacccccc
     6001 gctgctgccc ccgctgcttt caggtgttgc gctctcgctt gtgccaggtg tctcagagcc
     6061 agaggagcct ccgctagatc ctccggagtt gatggagctc tgggccttct tctgagcatt
     6121 gaacacctgt ctaggcatcc ggtagaagtc gcacagcagg gcggcacatt catctgccag
     6181 gattccctcg gtaatttcga cgcggtgatt catgccgggg tagttcagca cgttcatcag
     6241 ggagcctgcg gcgccccgtt tgctgttcct cacgccaaac accacgcggc cgatcctaga
     6301 gtggatcatg gcgccggcgc acatcacgca aggctcgaat gtcacgtaca gggtggcgtc
     6361 aatcagtctg tagttctgca tgaccaggcc gccctgtctc agggccataa tttcggcatg
     6421 ggctgttggg tcgtgcaggc cgatggctct gttccagccc tcgccgatca ctctattgtt
     6481 cagcaccagc acggctccca caggcacctc cctctcatcg cgtgccctct tggccagggt
     6541 cagggcatgt ctcatccagt actcgtggga aaactccacc tcagagccgc tgccgccacc
     6601 gctgcctccg ccgctaccgc cgactttcct cttcttcttg ggctcgaatt cgctgccgtc
     6661 ggcggttctt ttcatggtgg caccggtagt atgagttgcg tggccacgga gcctgaacag
     6721 catgatatgc gtagcatttc atagttacgg taaatattta agcctcatta aaacactcta
     6781 taacttcatg tgtactggct cccacatttt aaaacactta gaagaattgg taggctaaga
     6841 gtttttccaa aactacactg taaaaatcac aaatatttgg aagcagttgc ttttcccagt
     6901 ttgctcctga attctcatct acatagtacc gcctccattc tagagagggc ctatttccca
     6961 tgattccttc atatttgcat atacgataca aggctgttag agagataatt agaattaatt
     7021 tgactgtaaa cacaaagata ttagtacaaa atacgtgacg tagaaagtaa taatttcttg
     7081 ggtagtttgc agttttaaaa ttatgtttta aaatggacta tcatatgctt accgtaactt
     7141 gaaagtattt cgatttcttg gctttatata tcttgtggaa aggacgaaac accggaggac
     7201 cgccctgggc ctgggaggtt gtagctcccg aaacgttgct acaataaggc cgtctgaaaa
     7261 gatgtgccgc aacgctctgc cccttaaagc ttctgcttta aggggcatcg tttatttttt
     7321 tgtttaaact ctagaggccg caggaacccc tagtgatgga gttggccact ccctctctgc
     7381 gcgctcgctc gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggcggcctca
     7441 gtgagcgagc gagcgcgcag ctgcctgcag g
//
