LOCUS       sequence-388970-        7480 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV vector encoding a human codon-optimized 
            ecDHFR-enOsCas12f1-ecDHFR driven by MHCK7 promoter, DMD-targeting 
            guide RNAs compatible with enOsCas12f1 driven by hU6.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7480)
  AUTHORS   Kong X, Zhang H, Li G, Wang Z, Kong X, Wang L, Xue M, Zhang W, Wang 
            Y, Lin J, Zhou J, Shen X, Wei Y, Zhong N, Bai W, Yuan Y, Shi L, Zhou
            Y, Yang H
  TITLE     Engineered CRISPR-OsCas12f1 and RhCas12f1 with robust activities and
            expanded target range for genome editing.
  JOURNAL   Nat Commun. 2023 Apr 11;14(1):2046. doi: 10.1038/s41467-023-37829-7.
  PUBMED    37041195
REFERENCE   2  (bases 1 to 7480)
  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: "2023-04-11"; volume: "14(1):2046. doi"; pages: " 
            10.1038/s41467-023-37829-7"
COMMENT     Sequence Label: AAV2 ITR_phU6_gRNA scaffold-DMD sg1_phU6_gRNA
            ...
FEATURES             Location/Qualifiers
     source          1..7480
                     /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        143..391
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     promoter        562..810
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     regulatory      1770..1779
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1779..1844
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1851..1871
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1902..2375
                     /codon_start=1
                     /gene="folA"
                     /product="E. coli dihydrofolate reductase"
                     /label=ecDHFR
                     /translation="ISLIAALAVDYVIGMENAMPWNLPADLAWFKRNTLNKPVIMGRHT
                     WESIGRPLPGRKNIILSSQPSTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVIEQFLP
                     KAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILERR"
     CDS             2397..2417
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3717..3764
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             3765..4241
                     /codon_start=1
                     /gene="folA"
                     /product="E. coli dihydrofolate reductase"
                     /label=ecDHFR
                     /translation="ISLIAALAVDHVIGMETVMPWNLPADLAWFKRNTLNKPVIMGRHT
                     WESIGRPLPGRKNIILSSQPSTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVYEQFLP
                     KAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILERR"
     primer_bind     complement(4325..4345)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    4521..4728
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4743..4883
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4743..4872
                     /label=AAV2 ITR
     rep_origin      4958..5413
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5045..5064)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5255..5276
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5430..5449)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5549..5571
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5609..5627)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5695..5799
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5800..5868,5869..6660)
                     /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(6018..6037)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6831..7419
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7320..7339
                     /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 gagagggcct atttcccatg attccttcat atttgcatat
      181 acgatacaag gctgttagag agataattgg aattaatttg actgtaaaca caaagatatt
      241 agtacaaaat acgtgacgta gaaagtaata atttcttggg tagtttgcag ttttaaaatt
      301 atgttttaaa atggactatc atatgcttac cgtaacttga aagtatttcg atttcttggc
      361 tttatatatc ttgtggaaag gacgaaacac cgagggccga cttcccggcc caaaatcgag
      421 acagtagccg taaaacgttg agtttcagcg tgggcgacac actcgaaaag gttaagatat
      481 gcacatagta atccgtgcat gagccgcgaa agcggcttga aggattggct ttgatttccc
      541 tagttttatt tttttaagct tgagggccta tttcccatga ttccttcata tttgcatata
      601 cgatacaagg ctgttagaga gataattgga attaatttga ctgtaaacac aaagatatta
      661 gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta
      721 tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct
      781 ttatatatct tgtggaaagg acgaaacacc gagggccgac ttcccggccc aaaatcgaga
      841 cagtagccgt aaaacgttga gtttcagcgt gggcgacaca ctcgaaaagg ttaagatatg
      901 cacatagtaa tccgtgcatg agccgcgaaa gcggcttgaa ggtgtattca agctcaaggc
      961 ctttttattt ttttggtctc tttttttacc cttcagatta aaaataactg aggtaagggc
     1021 ctgggtaggg gaggtggtgt gagacgctcc tgtctctcct ctatctgccc atcggccctt
     1081 tggggaggag gaatgtgccc aaggactaaa aaaaggccat ggagccagag gggcgagggc
     1141 aacagacctt tcatgggcaa accttggggc cctgctgtct agcatgcccc actacgggtc
     1201 taggctgccc atgtaaggag gcaaggcctg gggacacccg agatgcctgg ttataattaa
     1261 cccagacatg tggctgcccc ccccccccca acacctgctg cctctaaaaa taaccctgtc
     1321 cctggtggat cccctgcatg cgaagatctt cgaacaaggc tgtgggggac tgagggcagg
     1381 ctgtaacagg cttgggggcc agggcttata cgtgcctggg actcccaaag tattactgtt
     1441 ccatgttccc ggcgaagggc cagctgtccc ccgccagcta gactcagcac ttagtttagg
     1501 aaccagtgag caagtcagcc cttggggcag cccatacaag gccatggggc tgggcaagct
     1561 gcacgcctgg gtccggggtg ggcacggtgc ccgggcaacg agctgaaagc tcatctgctc
     1621 tcaggggccc ctccctgggg acagcccctc ctggctagtc acaccctgta ggctcctcta
     1681 tataacccag gggcacaggg gctgccctca ttctaccacc acctccacag cacagacaga
     1741 cactcaggag ccagccaggt tggaccggtg ccaccatgga ctataaggac cacgacggag
     1801 actacaagga tcatgatatt gattacaaag acgatgacga taagatggcc ccaaagaaga
     1861 agcggaaggt cggtatccac ggagtcccag cagccggatc catcagtctg attgcggcgt
     1921 tagcggtaga ttacgttatc ggcatggaaa acgccatgcc gtggaacctg cctgccgatc
     1981 tcgcctggtt taaacgcaac accttaaata aacccgtgat tatgggccgc catacctggg
     2041 aatcaatcgg tcgtccgttg ccaggacgca aaaatattat cctcagcagt caaccgagta
     2101 cggacgatcg cgtaacgtgg gtgaagtcgg tggatgaagc catcgcggcg tgtggtgacg
     2161 taccagaaat catggtgatt ggcggcggtc gcgttattga acagttcttg ccaaaagcgc
     2221 aaaaactgta tctgacgcat atcgacgcag aagtggaagg cgacacccat ttcccggatt
     2281 acgagccgga tgactgggaa tcggtattca gcgaattcca cgatgctgat gcgcagaact
     2341 ctcacagcta ttgctttgag attctggagc ggcgaggaag cggaagcgga tccatgccta
     2401 agaagaagag aaaggtgatg ggaaagggcg tgctggccaa ggtgatgaaa tacgagctga
     2461 gatacctgga tggttgtggc gacttcagca atatgcagga gcaggtgtgg gccctgcagc
     2521 ggcagacacg ggaaatcctg aacagatcca tccaaatcgc cttccaatgg cgctgcgcca
     2581 acagcgagca ccacagaaag accggcgagt acctggacct gaaaacggaa accggctaca
     2641 agagactgga tggccacatc tacaactgcc tgaagggcca gtacgaggac atggccacat
     2701 ctaacctgaa cgccaccatc cagaaggctt ggaagaagta taactccagc aagaaggaaa
     2761 tcctgagggg cagcatgagc atccccagct acaagatgaa ccagcctctg cgactggaca
     2821 agaataccgt gaaactgtct gagggcgagc ggaacccaat cgtgaccctg acactgttta
     2881 gcgacaagtt caagcgggcc cagggcgtgt ccaacgtgaa gtttagcatg cctctgcacg
     2941 acggcaccca gagagccatc ttcgccaacc tgatgaacgg cacctaccag ctgggagagt
     3001 gccagctggt gtacaaacgg cctaagtggt tcctgttcgt gacatacaag ttcccccccg
     3061 tggaacatcc tctcgatcct gacaagattc tgggcgtcga catgggcgag gcctgcgcgc
     3121 tttatgcctc tacattcggc gagcacggct acctgaagat cgatggaggc gagattacaa
     3181 agtacgccaa gaagatggaa gctagaatcc ggagcatgca gaagcaggct gctcactgtg
     3241 gcgaaggcag aatcgggcac ggcaccaaaa caagagtgtc tgtggtgtac caggccaagg
     3301 acaaggtggc cagattcaga gataccatca accacagata ctctaaggcc ctgatcgact
     3361 acgccctgaa gaaccagtgt ggcaccatcc agatggaaga tctgaccggc atcaaggaag
     3421 atacaggatt tccaaagttc ctgagacatt ggacctacta cgacctgcag agcaagatcg
     3481 aggctaaggc cgccgagcac ggcatccaag ttgtcaagat caaccctaga cacaccagcc
     3541 agcgctgcag cagatgtgga cacatcgaca aagccaatag aaccagccaa gctgatttct
     3601 gctgcaccaa gtgcggcttc agcgccaatg ccgactttaa tgccagccag aacatcagca
     3661 tcagaaacat cgacaagatt atcgccaagg ctatcggcgc caaccggaag cagaccaaaa
     3721 ggccggcggc cacgaagaag gccggccagg caaagaagaa gaagatcagt ctgattgcgg
     3781 cgttagcggt agatcacgtt atcggcatgg aaaccgtcat gccgtggaac ctgcctgccg
     3841 atctcgcctg gtttaaacgc aacaccttaa ataaacccgt gattatgggc cgccatacct
     3901 gggaatcaat cggtcgtccg ttgccaggac gcaaaaatat tatcctcagc agtcaaccga
     3961 gtacggacga tcgcgtaacg tgggtgaagt cggtggatga agccatcgcg gcgtgtggtg
     4021 acgtaccaga aatcatggtt attggcggcg gtcgcgttta tgaacagttc ttgccaaaag
     4081 cgcaaaaact gtatctgacg catatcgacg cagaagtgga aggcgacacc catttcccgg
     4141 attacgagcc ggatgactgg gaatcggtat tcagcgaatt ccacgatgct gatgcgcaga
     4201 actctcacag ctattgcttt gagattctgg agcggcgata agaattccta gagctcgctg
     4261 atcagcctcg aaatcaacct ctggattaca aaatttgtga aagattgact ggtattctta
     4321 actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta
     4381 ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggtta gttcttgcca
     4441 cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg ctgttgggca
     4501 ctgacaattc cgtggtgtta ctgtgccttc tagttgccag ccatctgttg tttgcccctc
     4561 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga
     4621 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca
     4681 ggacagcaag ggggaggatt gggaagagaa tagcaggcat gctggggacg gccgagggcc
     4741 gcaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga
     4801 ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga
     4861 gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat tttctcctta cgcatctgtg
     4921 cggtatttca caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta
     4981 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccttagcg
     5041 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     5101 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     5161 aaaaaacttg atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     5221 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     5281 acactcaact ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggtc
     5341 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
     5401 acgtttacaa ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     5461 cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     5521 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     5581 tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat
     5641 gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga
     5701 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     5761 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     5821 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     5881 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     5941 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     6001 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     6061 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     6121 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     6181 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     6241 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     6301 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     6361 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     6421 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     6481 tttattgctg ataaatctgg agccggtgag cgtggaagcc gcggtatcat tgcagcactg
     6541 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     6601 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     6661 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     6721 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     6781 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     6841 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     6901 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     6961 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     7021 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     7081 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     7141 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     7201 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     7261 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     7321 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     7381 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     7441 ttacggttcc tggccttttg ctggcctttt gctcacatgt
//
