LOCUS       sequence-388968-        6433 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV vector encoding a human codon-optimized enOsCas12f1 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 6433)
  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 6433)
  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..6433
                     /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             1848..1868
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3168..3215
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(3278..3298)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    3474..3681
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3696..3836
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3696..3825
                     /label=AAV2 ITR
     rep_origin      3911..4366
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3998..4017)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4208..4229
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4383..4402)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4502..4524
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4562..4580)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4648..4752
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4753..4821,4822..5613)
                     /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(4971..4990)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5784..6372
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6273..6292
                     /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 taagatgcct aagaagaaga
     1861 gaaaggtgat gggaaagggc gtgctggcca aggtgatgaa atacgagctg agatacctgg
     1921 atggttgtgg cgacttcagc aatatgcagg agcaggtgtg ggccctgcag cggcagacac
     1981 gggaaatcct gaacagatcc atccaaatcg ccttccaatg gcgctgcgcc aacagcgagc
     2041 accacagaaa gaccggcgag tacctggacc tgaaaacgga aaccggctac aagagactgg
     2101 atggccacat ctacaactgc ctgaagggcc agtacgagga catggccaca tctaacctga
     2161 acgccaccat ccagaaggct tggaagaagt ataactccag caagaaggaa atcctgaggg
     2221 gcagcatgag catccccagc tacaagatga accagcctct gcgactggac aagaataccg
     2281 tgaaactgtc tgagggcgag cggaacccaa tcgtgaccct gacactgttt agcgacaagt
     2341 tcaagcgggc ccagggcgtg tccaacgtga agtttagcat gcctctgcac gacggcaccc
     2401 agagagccat cttcgccaac ctgatgaacg gcacctacca gctgggagag tgccagctgg
     2461 tgtacaaacg gcctaagtgg ttcctgttcg tgacatacaa gttccccccc gtggaacatc
     2521 ctctcgatcc tgacaagatt ctgggcgtcg acatgggcga ggcctgcgcg ctttatgcct
     2581 ctacattcgg cgagcacggc tacctgaaga tcgatggagg cgagattaca aagtacgcca
     2641 agaagatgga agctagaatc cggagcatgc agaagcaggc tgctcactgt ggcgaaggca
     2701 gaatcgggca cggcaccaaa acaagagtgt ctgtggtgta ccaggccaag gacaaggtgg
     2761 ccagattcag agataccatc aaccacagat actctaaggc cctgatcgac tacgccctga
     2821 agaaccagtg tggcaccatc cagatggaag atctgaccgg catcaaggaa gatacaggat
     2881 ttccaaagtt cctgagacat tggacctact acgacctgca gagcaagatc gaggctaagg
     2941 ccgccgagca cggcatccaa gttgtcaaga tcaaccctag acacaccagc cagcgctgca
     3001 gcagatgtgg acacatcgac aaagccaata gaaccagcca agctgatttc tgctgcacca
     3061 agtgcggctt cagcgccaat gccgacttta atgccagcca gaacatcagc atcagaaaca
     3121 tcgacaagat tatcgccaag gctatcggcg ccaaccggaa gcagaccaaa aggccggcgg
     3181 ccacgaagaa ggccggccag gcaaagaaga agaagtaaga attcaatcaa cctctggatt
     3241 acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg
     3301 gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct
     3361 cctccttgta taaatcctgg ttagttcttg ccacggcgga actcatcgcc gcctgccttg
     3421 cccgctgctg gacaggggct cggctgttgg gcactgacaa ttccgtggtg ttactgtgcc
     3481 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     3541 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     3601 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     3661 gaatagcagg catgctgggg acggccgagg gccgcaggaa cccctagtga tggagttggc
     3721 cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg
     3781 cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg
     3841 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag
     3901 caaccatagt acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     3961 agcgtgaccg ctacacttgc cagcgcctta gcgcccgctc ctttcgcttt cttcccttcc
     4021 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     4081 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgatttggg tgatggttca
     4141 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     4201 tttaatagtg gactcttgtt ccaaactgga acaacactca actctatctc gggctattct
     4261 tttgatttat aagggatttt gccgatttcg gtctattggt taaaaaatga gctgatttaa
     4321 caaaaattta acgcgaattt taacaaaata ttaacgttta caattttatg gtgcactctc
     4381 agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct
     4441 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     4501 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag
     4561 ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg
     4621 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     4681 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     4741 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     4801 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     4861 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     4921 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     4981 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     5041 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     5101 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     5161 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     5221 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     5281 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     5341 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     5401 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     5461 gagcgtggaa gccgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     5521 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     5581 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     5641 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     5701 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     5761 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     5821 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     5881 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
     5941 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     6001 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     6061 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     6121 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     6181 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     6241 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     6301 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     6361 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     6421 tttgctcaca tgt
//
