LOCUS       sequence-368938-        6744 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Npu Split N-terminal half of ABEmaxNGA with gRNA targeting mdx4cv 
            nonsense mutation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6744)
  AUTHORS   Xu L, Zhang C, Li H, Wang P, Gao Y, Mokadam NA, Ma J, Arnold WD, Han
            R
  TITLE     Efficient precise in vivo base editing in adult dystrophic mice.
  JOURNAL   Nat Commun. 2021 Jun 17;12(1):3719. doi: 10.1038/s41467-021-23996-y.
  PUBMED    34140489
REFERENCE   2  (bases 1 to 6744)
  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: "2021-06-17"; volume: "12(1):3719. doi"; pages: " 
            10.1038/s41467-021-23996-y"
COMMENT     Sequence Label: pX601‐mhCMV‐miniABEmax‐N3‐ E53ogRNA
FEATURES             Location/Qualifiers
     source          1..6744
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        566..665
                     /label=CMVd1 promoter
                     /note="truncated human cytomegalovirus (CMV) immediate 
                     early promoter that yields slightly reduced expression 
                     relative to the full-length promoter"
     primer_bind     616..636
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      688..697
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             739..759
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1360..3075
                     /codon_start=1
                     /product="N-terminal portion of Streptococcus pyogenes Cas9
                     (Zetsche et al., 2015)"
                     /label=Cas9(N)
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIE"
     primer_bind     complement(3415..3432)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3421..3628
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        3636..3884
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     3636..3656
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3807..3826
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     repeat_region   4007..4147
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4007..4136
                     /label=AAV2 ITR
     rep_origin      4222..4677
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4309..4328)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4519..4540
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4694..4713)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4813..4835
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4873..4891)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4959..5063
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5064..5132,5133..5924)
                     /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(5282..5301)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6095..6683
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6584..6603
                     /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 gactcgaggc ccttcagatt aaaaataact gaggtaaggg
      181 cctgggtagg ggaggtggtg tgagacgctc ctgtctctcc tctatctgcc catcggccct
      241 ttggggagga ggaatgtgcc caaggactaa aaaaaggcca tggagccaga ggggcgaggg
      301 caacagacct ttcatgggca aaccttgggg ccctgctgac tgtagatgag agcagccact
      361 acgggtctag gctgcccatg taaggaggca aggcctgggg acacccgaga tgcctggtta
      421 taattaaccc agacatgtgg ctgccccccc cccccaacac ctgctgcctg ctaaaaataa
      481 ccctgtccct ggtggccctg catgcccact cacggggatt tccaagtctc caccccattg
      541 acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca
      601 actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca
      661 gagctggttt agtgaaccgt cagatccgcc accatggcta gcatgaaacg gacagccgac
      721 ggaagcgagt tcgagtcacc aaagaagaag cggaaagtcg gatcctctga ggtggagttt
      781 tcccacgagt actggatgag acatgccctg accctggcca agagggcacg cgatgagagg
      841 gaggtgcctg tgggagccgt gctggtgctg aacaatagag tgatcggcga gggctggaac
      901 agagccatcg gcctgcacga cccaacaggc catgccgaaa ttatggccct gaggcagggc
      961 ggcctggtca tgcagaacta cagactgatt gacgccaccc tgtacgggac attcgagcct
     1021 tgcgtgatgt gcgccggcgc catgatccac tctaggatcg gccgcgtggt gtttggcgtg
     1081 aggaacgcaa aaaccggcgc cgcaggctcc ctgatggacg tgctgcacta ccccggcatg
     1141 aatcaccgcg tcgaaattac cgagggaatc ctggcagatg aatgtgccgc cctgctgtgc
     1201 tatttctttc ggatgcctag acaggtgttc aatgctcaga agaaggccca gagctccacc
     1261 gactccggag gatctagcgg aggctcctct ggctctgaga cacctggcac aagcgagagc
     1321 gcaacacctg aaagcagcgg gggcagcagc ggggggtcag acaagaagta cagcatcggc
     1381 ctggccatcg gcaccaactc tgtgggctgg gccgtgatca ccgacgagta caaggtgccc
     1441 agcaagaaat tcaaggtgct gggcaacacc gaccggcaca gcatcaagaa gaacctgatc
     1501 ggagccctgc tgttcgacag cggcgaaaca gccgaggcca cccggctgaa gagaaccgcc
     1561 agaagaagat acaccagacg gaagaaccgg atctgctatc tgcaagagat cttcagcaac
     1621 gagatggcca aggtggacga cagcttcttc cacagactgg aagagtcctt cctggtggaa
     1681 gaggataaga agcacgagcg gcaccccatc ttcggcaaca tcgtggacga ggtggcctac
     1741 cacgagaagt accccaccat ctaccacctg agaaagaaac tggtggacag caccgacaag
     1801 gccgacctgc ggctgatcta tctggccctg gcccacatga tcaagttccg gggccacttc
     1861 ctgatcgagg gcgacctgaa ccccgacaac agcgacgtgg acaagctgtt catccagctg
     1921 gtgcagacct acaaccagct gttcgaggaa aaccccatca acgccagcgg cgtggacgcc
     1981 aaggccatcc tgtctgccag actgagcaag agcagacggc tggaaaatct gatcgcccag
     2041 ctgcccggcg agaagaagaa tggcctgttc ggaaacctga ttgccctgag cctgggcctg
     2101 acccccaact tcaagagcaa cttcgacctg gccgaggatg ccaaactgca gctgagcaag
     2161 gacacctacg acgacgacct ggacaacctg ctggcccaga tcggcgacca gtacgccgac
     2221 ctgtttctgg ccgccaagaa cctgtccgac gccatcctgc tgagcgacat cctgagagtg
     2281 aacaccgaga tcaccaaggc ccccctgagc gcctctatga tcaagagata cgacgagcac
     2341 caccaggacc tgaccctgct gaaagctctc gtgcggcagc agctgcctga gaagtacaaa
     2401 gagattttct tcgaccagag caagaacggc tacgccggct acattgacgg cggagccagc
     2461 caggaagagt tctacaagtt catcaagccc atcctggaaa agatggacgg caccgaggaa
     2521 ctgctcgtga agctgaacag agaggacctg ctgcggaagc agcggacctt cgacaacggc
     2581 agcatccccc accagatcca cctgggagag ctgcacgcca ttctgcggcg gcaggaagat
     2641 ttttacccat tcctgaagga caaccgggaa aagatcgaga agatcctgac cttccgcatc
     2701 ccctactacg tgggccctct ggccagggga aacagcagat tcgcctggat gaccagaaag
     2761 agcgaggaaa ccatcacccc ctggaacttc gaggaagtgg tggacaaggg cgcttccgcc
     2821 cagagcttca tcgagcggat gaccaacttc gataagaacc tgcccaacga gaaggtgctg
     2881 cccaagcaca gcctgctgta cgagtacttc accgtgtata acgagctgac caaagtgaaa
     2941 tacgtgaccg agggaatgag aaagcccgcc ttcctgagcg gcgagcagaa aaaggccatc
     3001 gtggacctgc tgttcaagac caaccggaaa gtgaccgtga agcagctgaa agaggactac
     3061 ttcaagaaaa tcgagtgcct gtcctacgag acagagatcc tgacagtgga gtatggcctg
     3121 ctgccaatcg gcaagatcgt ggagaagagg atcgagtgta ccgtgtactc tgtggataac
     3181 aatggcaaca tctatacaca gcccgtggca cagtggcacg ataggggaga gcaggaggtg
     3241 ttcgagtatt gcctggagga cggcagcctg atcagggcaa ccaaggacca caagttcatg
     3301 acagtggatg gccagatgct gcccatcgac gagattttcg agcgggagct ggacctgatg
     3361 agagtggata acctgcctaa ttaactagta gaattcctag agctcgctga tcagcctcga
     3421 ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc
     3481 tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc
     3541 tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt
     3601 gggaagagaa tagcaggcat gctggggagg tacctgaggg cctatttccc atgattcctt
     3661 catatttgca tatacgatac aaggctgtta gagagataat tggaattaat ttgactgtaa
     3721 acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg
     3781 cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt
     3841 tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccggttat ctcctgttct
     3901 gcagcgtttc agagctatgc tggaaacagc atagcaagtt gaaataaggc tagtccgtta
     3961 tcaacttgaa aaagtggcac cgagtcggtg ctttttttgc ggccgcagga acccctagtg
     4021 atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag
     4081 gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc
     4141 ctgcaggggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     4201 atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     4261 tggttacgcg cagcgtgacc gctacacttg ccagcgcctt agcgcccgct cctttcgctt
     4321 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     4381 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgatttgg
     4441 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     4501 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aactctatct
     4561 cgggctattc ttttgattta taagggattt tgccgatttc ggtctattgg ttaaaaaatg
     4621 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaattttat
     4681 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc
     4741 caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag
     4801 ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg
     4861 cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg
     4921 tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     4981 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     5041 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     5101 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     5161 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     5221 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     5281 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca
     5341 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     5401 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     5461 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     5521 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     5581 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa
     5641 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     5701 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     5761 ctggagccgg tgagcgtgga agccgcggta tcattgcagc actggggcca gatggtaagc
     5821 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     5881 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     5941 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     6001 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     6061 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     6121 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     6181 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     6241 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     6301 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     6361 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     6421 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     6481 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     6541 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     6601 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     6661 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     6721 tttgctggcc ttttgctcac atgt
//
