LOCUS       sequence-369021-        6616 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Npu Split C-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 6616)
  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 6616)
  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‐ABEmaxNGA‐C3‐ E53ogRNA
FEATURES             Location/Qualifiers
     source          1..6616
                     /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"
     CDS             806..3190
                     /codon_start=1
                     /product="C-terminal portion of Streptococcus pyogenes Cas9
                     (Zetsche et al., 2015)"
                     /label=Cas9(C)
                     /translation="CFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILED
                     IVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGK
                     TILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKG
                     ILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQI
                     LKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDN
                     KVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDK
                     AGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQF
                     YKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGK
                     ATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMP
                     QVNIVKKTEVQTGGFSKESIRPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVVAKV
                     EKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELEN
                     GRKRMLASARFLQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLD
                     EIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPRAFKY
                     FDTTIDRKQYRSTKEVLDATLIHQSITGLYETRIDLSQLGGD"
     CDS             3233..3253
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(3287..3304)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3293..3500
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        3508..3756
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     3508..3528
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3679..3698
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     repeat_region   3879..4019
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3879..4008
                     /label=AAV2 ITR
     rep_origin      4094..4549
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4181..4200)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4391..4412
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4566..4585)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4685..4707
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4745..4763)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4831..4935
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4936..5004,5005..5796)
                     /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(5154..5173)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5967..6555
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6456..6475
                     /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 cagatccgct agccaccatg atcaagattg ctacacggaa
      721 atacctggga aagcagaacg tgtacgacat cggcgtggag cgggatcaca acttcgccct
      781 gaagaatggc tttatcgcca gcaattgctt cgactccgtg gaaatctccg gcgtggaaga
      841 tcggttcaac gcctccctgg gcacatacca cgatctgctg aaaattatca aggacaagga
      901 cttcctggac aatgaggaaa acgaggacat tctggaagat atcgtgctga ccctgacact
      961 gtttgaggac agagagatga tcgaggaacg gctgaaaacc tatgcccacc tgttcgacga
     1021 caaagtgatg aagcagctga agcggcggag atacaccggc tggggcaggc tgagccggaa
     1081 gctgatcaac ggcatccggg acaagcagtc cggcaagaca atcctggatt tcctgaagtc
     1141 cgacggcttc gccaacagaa acttcatgca gctgatccac gacgacagcc tgacctttaa
     1201 agaggacatc cagaaagccc aggtgtccgg ccagggcgat agcctgcacg agcacattgc
     1261 caatctggcc ggcagccccg ccattaagaa gggcatcctg cagacagtga aggtggtgga
     1321 cgagctcgtg aaagtgatgg gccggcacaa gcccgagaac atcgtgatcg aaatggccag
     1381 agagaaccag accacccaga agggacagaa gaacagccgc gagagaatga agcggatcga
     1441 agagggcatc aaagagctgg gcagccagat cctgaaagaa caccccgtgg aaaacaccca
     1501 gctgcagaac gagaagctgt acctgtacta cctgcagaat gggcgggata tgtacgtgga
     1561 ccaggaactg gacatcaacc ggctgtccga ctacgatgtg gaccatatcg tgcctcagag
     1621 ctttctgaag gacgactcca tcgacaacaa ggtgctgacc agaagcgaca agaaccgggg
     1681 caagagcgac aacgtgccct ccgaagaggt cgtgaagaag atgaagaact actggcggca
     1741 gctgctgaac gccaagctga ttacccagag aaagttcgac aatctgacca aggccgagag
     1801 aggcggcctg agcgaactgg ataaggccgg cttcatcaag agacagctgg tggaaacccg
     1861 gcagatcaca aagcacgtgg cacagatcct ggactcccgg atgaacacta agtacgacga
     1921 gaatgacaag ctgatccggg aagtgaaagt gatcaccctg aagtccaagc tggtgtccga
     1981 tttccggaag gatttccagt tttacaaagt gcgcgagatc aacaactacc accacgccca
     2041 cgacgcctac ctgaacgccg tcgtgggaac cgccctgatc aaaaagtacc ctaagctgga
     2101 aagcgagttc gtgtacggcg actacaaggt gtacgacgtg cggaagatga tcgccaagag
     2161 cgagcaggaa atcggcaagg ctaccgccaa gtacttcttc tacagcaaca tcatgaactt
     2221 tttcaagacc gagattaccc tggccaacgg cgagatccgg aagcggcctc tgatcgagac
     2281 aaacggcgaa accggggaga tcgtgtggga taagggccgg gattttgcca ccgtgcggaa
     2341 agtgctgagc atgccccaag tgaatatcgt gaaaaagacc gaggtgcaga caggcggctt
     2401 cagcaaagag tctatccggc ccaagaggaa cagcgataag ctgatcgcca gaaagaagga
     2461 ctgggaccct aagaagtacg gcggcttcgt cagccccacc gtggcctatt ctgtgctggt
     2521 ggtggccaaa gtggaaaagg gcaagtccaa gaaactgaag agtgtgaaag agctgctggg
     2581 gatcaccatc atggaaagaa gcagcttcga gaagaatccc atcgactttc tggaagccaa
     2641 gggctacaaa gaagtgaaaa aggacctgat catcaagctg cctaagtact ccctgttcga
     2701 gctggaaaac ggccggaaga gaatgctggc ctctgcccgc tttctgcaga agggaaacga
     2761 actggccctg ccctccaaat atgtgaactt cctgtacctg gccagccact atgagaagct
     2821 gaagggctcc cccgaggata atgagcagaa acagctgttt gtggaacagc acaagcacta
     2881 cctggacgag atcatcgagc agatcagcga gttctccaag agagtgatcc tggccgacgc
     2941 taatctggac aaagtgctgt ccgcctacaa caagcaccgg gataagccca tcagagagca
     3001 ggccgagaat atcatccacc tgtttaccct gaccaatctg ggagcccctc gggccttcaa
     3061 gtactttgac accaccatcg accggaagca gtaccggagc accaaagagg tgctggacgc
     3121 caccctgatc caccagagca tcaccggcct gtacgagaca cggatcgacc tgtctcagct
     3181 gggaggtgac tctggcggct caaaaagaac cgccgacggc agcgaattcg agcccaagaa
     3241 gaagaggaaa gtctaactag tagaattcct agagctcgct gatcagcctc gactgtgcct
     3301 tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt
     3361 gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg
     3421 tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagag
     3481 aatagcaggc atgctgggga ggtacctgag ggcctatttc ccatgattcc ttcatatttg
     3541 catatacgat acaaggctgt tagagagata attggaatta atttgactgt aaacacaaag
     3601 atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt tgcagtttta
     3661 aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta tttcgatttc
     3721 ttggctttat atatcttgtg gaaaggacga aacaccggtt atctcctgtt ctgcagcgtt
     3781 tcagagctat gctggaaaca gcatagcaag ttgaaataag gctagtccgt tatcaacttg
     3841 aaaaagtggc accgagtcgg tgcttttttt gcggccgcag gaacccctag tgatggagtt
     3901 ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg
     3961 acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcagct gcctgcaggg
     4021 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatacgtca
     4081 aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     4141 cgcagcgtga ccgctacact tgccagcgcc ttagcgcccg ctcctttcgc tttcttccct
     4201 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     4261 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgattt gggtgatggt
     4321 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     4381 ttctttaata gtggactctt gttccaaact ggaacaacac tcaactctat ctcgggctat
     4441 tcttttgatt tataagggat tttgccgatt tcggtctatt ggttaaaaaa tgagctgatt
     4501 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt atggtgcact
     4561 ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc
     4621 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
     4681 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga
     4741 aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag
     4801 acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     4861 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     4921 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     4981 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     5041 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     5101 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     5161 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     5221 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     5281 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     5341 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     5401 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     5461 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     5521 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     5581 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     5641 ggtgagcgtg gaagccgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     5701 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     5761 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     5821 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     5881 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     5941 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     6001 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     6061 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
     6121 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     6181 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     6241 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     6301 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     6361 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     6421 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     6481 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     6541 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     6601 ccttttgctc acatgt
//
