LOCUS       sequence-404750-        5747 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression vector of crRNA (DMD#20) targeting the dystrophin intron 
            44 just upstream of exon 45, and crRNA(DMD#23)targeting the 
            dystrophin intron 55 just downstream of exon 55. Blasticidin 
            selectable..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5747)
  AUTHORS   Kita Y, Okuzaki Y, Naoe Y, Lee J, Bang U, Okawa N, Ichiki A, 
            Jonouchi T, Sakurai H, Kojima Y, Hotta A
  TITLE     Dual CRISPR-Cas3 system for inducing multi-exon skipping in DMD 
            patient-derived iPSCs.
  JOURNAL   Stem Cell Reports. 2023 Sep 12;18(9):1753-1765. doi: 
            10.1016/j.stemcr.2023.07.007. Epub 2023 Aug 24.
  PUBMED    37625413
REFERENCE   2  (bases 1 to 5747)
  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"; doi: 
            "10.1016/j.stemcr.2023.07.007"; journalName: "Stem Cell Reports"; 
            date: "2023-09-12- 12"; volume: "18"; issue: "9"; pages: "1753-1765"
COMMENT     Sequence Label: pPV-C1-crRNA(DMD#20_DMD#23)-EF1a-BA
FEATURES             Location/Qualifiers
     source          1..5747
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   46..108
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     468..484
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     468..484
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(831..850)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        1120..2298
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          1351..2289
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     2246..2266
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    2321..2345
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      2374..2383
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2380..2778
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISQDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     primer_bind     complement(2891..2910)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2956..3011
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3010..3029)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     repeat_region   3714..3748
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(3841..3858)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4012..4600)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4092..4111)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4771..5562,5563..5631))
                     /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     5394..5413
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5632..5736)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gaaaagtgcc acctgacgtc atctgttaac attatacgcg tttaacccta gaaagataat
       61 catattgtga cgtacgttaa agataatcat gcgtaaaatt gacgcatgtg ttttatcggt
      121 ctgtatatcg aggtttattt attaatttga atagatatta agttttatta tatttacact
      181 tacatactaa taataaattc aacaaacaat ttatttatgt ttatttattt attaaaaaaa
      241 aacaaaaact caaaatttct tctataaagt aacaaaactt ttaaacattc tctcttttac
      301 aaaaataaac ttattttgta ctttaaaaac agtcatgttg tattataaaa taagtaatta
      361 gcttaaccta tacataatag aaacaaatta tacttattag tcagtcagaa acaactttgg
      421 cacatatcaa tattatgctc tgctagcgtt taaacccggg cgatcgctct agaactagtg
      481 gatcaatgat acggcgacca ccgagatctc cccgggctgc aggaattcaa aaaacggttt
      541 atccccgctg gcgcggggaa cacccttgta ctacagaata tgattctcac caagacggtt
      601 tatccccgct ggcgcgggga acactttagg aagcatgtac tggggtctac tgtcagcggt
      661 ttatccccgc tggcgcgggg aacactctaa acataaccta ttattaatta atgatttttt
      721 aagccagtca caatctacca actttatagt atcacacaaa caacacatcc acggtgtttc
      781 gtcctttcca caagatatat aaagccaaga aatcgaaata ctttcaagtt acggtaagca
      841 tatgatagtc cattttaaaa cataatttta aaactgcaaa ctacccaaga aattattact
      901 ttctacgtca cgtattttgt actaatatct ttgtgtttac agtcaaatta attccaatta
      961 tctctctaac agccttgtat cgtatatgca aatatgaagg aatcatggga agaattcacg
     1021 tcaattcgat atcaagcttt gcaaagatgg ataaagtttt aaacagagag gaatctttgc
     1081 agctaatgga ccttctaggt cttgaaagga gtgggaattg gctccggtgc ccgtcagtgg
     1141 gcagagcgca catcgcccac agtccccgag aagttggggg gaggggtcgg caattgaacc
     1201 ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta ctggctccgc
     1261 ctttttcccg agggtggggg agaaccgtat ataagtgcag tagtcgccgt gaacgttctt
     1321 tttcgcaacg ggtttgccgc cagaacacag gtaagtgccg tgtgtggttc ccgcgggcct
     1381 ggcctcttta cgggttatgg cccttgcgtg ccttgaatta cttccacctg gctgcagtac
     1441 gtgattcttg atcccgagct tcgggttgga agtgggtggg agagttcgag gccttgcgct
     1501 taaggagccc cttcgcctcg tgcttgagtt gaggcctggc ctgggcgctg gggccgccgc
     1561 gtgcgaatct ggtggcacct tcgcgcctgt ctcgctgctt tcgataagtc tctagccatt
     1621 taaaattttt gatgacctgc tgcgacgctt tttttctggc aagatagtct tgtaaatgcg
     1681 ggccaagatc tgcacactgg tatttcggtt tttggggccg cgggcggcga cggggcccgt
     1741 gcgtcccagc gcacatgttc ggcgaggcgg ggcctgcgag cgcggccacc gagaatcgga
     1801 cgggggtagt ctcaagctgg ccggcctgct ctggtgcctg gcctcgcgcc gccgtgtatc
     1861 gccccgccct gggcggcaag gctggcccgg tcggcaccag ttgcgtgagc ggaaagatgg
     1921 ccgcttcccg gccctgctgc agggagctca aaatggagga cgcggcgctc gggagagcgg
     1981 gcgggtgagt cacccacaca aaggaaaagg gcctttccgt cctcagccgt cgcttcatgt
     2041 gactccacgg agtaccgggc gccgtccagg cacctcgatt agttctcgag cttttggagt
     2101 acgtcgtctt taggttgggg ggaggggttt tatgcgatgg agtttcccca cactgagtgg
     2161 gtggagactg aagttaggcc agcttggcac ttgatgtaat tctccttgga atttgccctt
     2221 tttgagtttg gatcttggtt cattctcaag cctcagacag tggttcaaag tttttttctt
     2281 ccatttcagg tgtcgtgagg aattagcggt ggcggccatc acaagtttgt acaaaaaagc
     2341 aggctggcgc cggaaccaat tcaggaattc gccgccacca tggccaagcc tttgtctcaa
     2401 gaagaatcca ccctcattga aagagcaacg gctacaatca acagcatccc catctctcaa
     2461 gactacagcg tcgccagcgc agctctctct agcgacggcc gcatcttcac tggtgtcaat
     2521 gtatatcatt ttactggggg accttgtgca gaactcgtgg tgctgggcac tgctgctgct
     2581 gcggcagctg gcaacctgac ttgtatcgtc gcgatcggaa atgagaacag gggcatcttg
     2641 agcccctgcg gacggtgccg acaggtgctt ctcgatctgc atcctgggat caaagccata
     2701 gtgaaggaca gtgatggaca gccgacggca gttgggattc gtgaattgct gccctctggt
     2761 tatgtgtggg agggctaagg atcctaggct taagtatacc tcgagttaaa ttcactcctc
     2821 aggtgcaggc tgcctatcag aaggtggtgg ctggtgtggc caatgccctg gctcacaaat
     2881 accactgaga tctttttccc tctgccaaaa attatgggga catcatgaag ccccttgagc
     2941 atctgacttc tggctaataa aggaaattta ttttcattgc aatagtgtgt tggaattttt
     3001 tgtgtctctc actcggaagg acatatggga gggcaaatca tttaaaacat cagaatgagt
     3061 atttggttta gagtttggca acatatgccc atatgctggc tgccatgaac aaaggttggc
     3121 tataaagagg tcatcagtat atgaaacagc cccctgctgt ccattcctta ttccatagaa
     3181 aagccttgac ttgaggttag atttttttta tattttgttt tgtgttattt ttttctttaa
     3241 catccctaaa attttcctta catgttttac tagccagatt tttcctcctc tcctgactac
     3301 tcccagtcat agctgtccct cttctcttat ggagatccct cgacttaatt aaccaacaag
     3361 ctcgtcatcg ctttgcagaa gagcagagag gatatgctca tcgtctaaag aactacccat
     3421 tttattatat attagtcacg atatctataa caagaaaata tatatataat aagttatcac
     3481 gtaagtagaa catgaaataa caatataatt atcgtatgag ttaaatctta aaagtcacgt
     3541 aaaagataat catgcgtcat tttgactcac gcggtcgtta tagttcaaaa tcagtgacac
     3601 ttaccgcatt gacaagcacg cctcacggga gctccaagcg gcgactgaga tgtcctaaat
     3661 gcacagcgac ggattcgcgc tatttagaaa gagagagcaa tatttcaaga atgcatgcgt
     3721 caattttacg cagactatct ttctagggtt aatacgtata atacatatga ttcagctgca
     3781 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     3841 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     3901 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     3961 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     4021 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     4081 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     4141 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     4201 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     4261 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     4321 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     4381 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     4441 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     4501 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     4561 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     4621 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     4681 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     4741 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     4801 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     4861 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     4921 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     4981 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     5041 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     5101 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     5161 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     5221 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     5281 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     5341 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     5401 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     5461 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     5521 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     5581 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     5641 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     5701 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttcccc
//
