LOCUS       sequence-368934-        6634 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gp41-1 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 6634)
  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 6634)
  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‐C2‐ E53ogRNA
FEATURES             Location/Qualifiers
     source          1..6634
                     /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             824..3208
                     /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             3251..3271
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(3305..3322)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3311..3518
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        3526..3774
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     3526..3546
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3697..3716
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     repeat_region   3897..4037
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3897..4026
                     /label=AAV2 ITR
     rep_origin      4112..4567
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4199..4218)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4409..4430
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4584..4603)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4703..4725
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4763..4781)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4849..4953
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4954..5022,5023..5814)
                     /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(5172..5191)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5985..6573
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6474..6493
                     /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 atgctcaaga agatcctcaa
      721 gattgaagag ttggacgagc gcgagcttat agacatagaa gtcagtggta atcacctttt
      781 ctacgcaaat gacattttga ctcacaactc ctcttcagac gtttgcttcg actccgtgga
      841 aatctccggc gtggaagatc ggttcaacgc ctccctgggc acataccacg atctgctgaa
      901 aattatcaag gacaaggact tcctggacaa tgaggaaaac gaggacattc tggaagatat
      961 cgtgctgacc ctgacactgt ttgaggacag agagatgatc gaggaacggc tgaaaaccta
     1021 tgcccacctg ttcgacgaca aagtgatgaa gcagctgaag cggcggagat acaccggctg
     1081 gggcaggctg agccggaagc tgatcaacgg catccgggac aagcagtccg gcaagacaat
     1141 cctggatttc ctgaagtccg acggcttcgc caacagaaac ttcatgcagc tgatccacga
     1201 cgacagcctg acctttaaag aggacatcca gaaagcccag gtgtccggcc agggcgatag
     1261 cctgcacgag cacattgcca atctggccgg cagccccgcc attaagaagg gcatcctgca
     1321 gacagtgaag gtggtggacg agctcgtgaa agtgatgggc cggcacaagc ccgagaacat
     1381 cgtgatcgaa atggccagag agaaccagac cacccagaag ggacagaaga acagccgcga
     1441 gagaatgaag cggatcgaag agggcatcaa agagctgggc agccagatcc tgaaagaaca
     1501 ccccgtggaa aacacccagc tgcagaacga gaagctgtac ctgtactacc tgcagaatgg
     1561 gcgggatatg tacgtggacc aggaactgga catcaaccgg ctgtccgact acgatgtgga
     1621 ccatatcgtg cctcagagct ttctgaagga cgactccatc gacaacaagg tgctgaccag
     1681 aagcgacaag aaccggggca agagcgacaa cgtgccctcc gaagaggtcg tgaagaagat
     1741 gaagaactac tggcggcagc tgctgaacgc caagctgatt acccagagaa agttcgacaa
     1801 tctgaccaag gccgagagag gcggcctgag cgaactggat aaggccggct tcatcaagag
     1861 acagctggtg gaaacccggc agatcacaaa gcacgtggca cagatcctgg actcccggat
     1921 gaacactaag tacgacgaga atgacaagct gatccgggaa gtgaaagtga tcaccctgaa
     1981 gtccaagctg gtgtccgatt tccggaagga tttccagttt tacaaagtgc gcgagatcaa
     2041 caactaccac cacgcccacg acgcctacct gaacgccgtc gtgggaaccg ccctgatcaa
     2101 aaagtaccct aagctggaaa gcgagttcgt gtacggcgac tacaaggtgt acgacgtgcg
     2161 gaagatgatc gccaagagcg agcaggaaat cggcaaggct accgccaagt acttcttcta
     2221 cagcaacatc atgaactttt tcaagaccga gattaccctg gccaacggcg agatccggaa
     2281 gcggcctctg atcgagacaa acggcgaaac cggggagatc gtgtgggata agggccggga
     2341 ttttgccacc gtgcggaaag tgctgagcat gccccaagtg aatatcgtga aaaagaccga
     2401 ggtgcagaca ggcggcttca gcaaagagtc tatccggccc aagaggaaca gcgataagct
     2461 gatcgccaga aagaaggact gggaccctaa gaagtacggc ggcttcgtca gccccaccgt
     2521 ggcctattct gtgctggtgg tggccaaagt ggaaaagggc aagtccaaga aactgaagag
     2581 tgtgaaagag ctgctgggga tcaccatcat ggaaagaagc agcttcgaga agaatcccat
     2641 cgactttctg gaagccaagg gctacaaaga agtgaaaaag gacctgatca tcaagctgcc
     2701 taagtactcc ctgttcgagc tggaaaacgg ccggaagaga atgctggcct ctgcccgctt
     2761 tctgcagaag ggaaacgaac tggccctgcc ctccaaatat gtgaacttcc tgtacctggc
     2821 cagccactat gagaagctga agggctcccc cgaggataat gagcagaaac agctgtttgt
     2881 ggaacagcac aagcactacc tggacgagat catcgagcag atcagcgagt tctccaagag
     2941 agtgatcctg gccgacgcta atctggacaa agtgctgtcc gcctacaaca agcaccggga
     3001 taagcccatc agagagcagg ccgagaatat catccacctg tttaccctga ccaatctggg
     3061 agcccctcgg gccttcaagt actttgacac caccatcgac cggaagcagt accggagcac
     3121 caaagaggtg ctggacgcca ccctgatcca ccagagcatc accggcctgt acgagacacg
     3181 gatcgacctg tctcagctgg gaggtgactc tggcggctca aaaagaaccg ccgacggcag
     3241 cgaattcgag cccaagaaga agaggaaagt ctaactagta gaattcctag agctcgctga
     3301 tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct
     3361 tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca
     3421 tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag
     3481 ggggaggatt gggaagagaa tagcaggcat gctggggagg tacctgaggg cctatttccc
     3541 atgattcctt catatttgca tatacgatac aaggctgtta gagagataat tggaattaat
     3601 ttgactgtaa acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt
     3661 gggtagtttg cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact
     3721 tgaaagtatt tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccggttat
     3781 ctcctgttct gcagcgtttc agagctatgc tggaaacagc atagcaagtt gaaataaggc
     3841 tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg ctttttttgc ggccgcagga
     3901 acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg
     3961 gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc
     4021 gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     4081 ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg
     4141 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgcctt agcgcccgct
     4201 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     4261 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     4321 cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     4381 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     4441 aactctatct cgggctattc ttttgattta taagggattt tgccgatttc ggtctattgg
     4501 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt
     4561 acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc
     4621 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     4681 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     4741 ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg
     4801 ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct
     4861 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     4921 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     4981 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     5041 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     5101 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     5161 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
     5221 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     5281 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     5341 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     5401 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     5461 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
     5521 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     5581 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     5641 gctgataaat ctggagccgg tgagcgtgga agccgcggta tcattgcagc actggggcca
     5701 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     5761 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     5821 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     5881 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     5941 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     6001 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     6061 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     6121 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     6181 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     6241 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     6301 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     6361 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     6421 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     6481 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     6541 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     6601 ttcctggcct tttgctggcc ttttgctcac atgt
//
