LOCUS       sequence-404745-        8196 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Single-strand annealing (SSA) vector for monitoring crRNA(DMDex45)'s
            activity. DMD intron 44 region (upstream of exon 45).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8196)
  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 8196)
  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-EF1a-EGxxFP(DMDex45_10)-iP-A
FEATURES             Location/Qualifiers
     source          1..8196
                     /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)"
     promoter        578..1756
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          809..1747
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1704..1724
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    1779..1803
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      1856..1865
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1862..1864,1865..1867,1868..2380)
                     /codon_start=1
                     /product="N-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VN173
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKTRHNIE"
     primer_bind     complement(1907..1928)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     3809..3830
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(3900..3924)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    join(3966..4540,4541..4543,4544..4552)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4133..4150)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4360..4379
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4553..5152
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(4553..4572)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5049..5069
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(5340..5359)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    5405..5460
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(5459..5478)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     repeat_region   6163..6197
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(6290..6307)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6461..7049)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6541..6560)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7220..8011,8012..8080))
                     /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     7843..7862
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8081..8185)
                     /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 agactagtgc
      481 ggccgctgca gaagctttgc aaagatggat aaagttttaa acagagagga atctttgcag
      541 ctaatggacc ttctaggtct tgaaaggagt gggaattggc tccggtgccc gtcagtgggc
      601 agagcgcaca tcgcccacag tccccgagaa gttgggggga ggggtcggca attgaaccgg
      661 tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct
      721 ttttcccgag ggtgggggag aaccgtatat aagtgcagta gtcgccgtga acgttctttt
      781 tcgcaacggg tttgccgcca gaacacaggt aagtgccgtg tgtggttccc gcgggcctgg
      841 cctctttacg ggttatggcc cttgcgtgcc ttgaattact tccacctggc tgcagtacgt
      901 gattcttgat cccgagcttc gggttggaag tgggtgggag agttcgaggc cttgcgctta
      961 aggagcccct tcgcctcgtg cttgagttga ggcctggcct gggcgctggg gccgccgcgt
     1021 gcgaatctgg tggcaccttc gcgcctgtct cgctgctttc gataagtctc tagccattta
     1081 aaatttttga tgacctgctg cgacgctttt tttctggcaa gatagtcttg taaatgcggg
     1141 ccaagatctg cacactggta tttcggtttt tggggccgcg ggcggcgacg gggcccgtgc
     1201 gtcccagcgc acatgttcgg cgaggcgggg cctgcgagcg cggccaccga gaatcggacg
     1261 ggggtagtct caagctggcc ggcctgctct ggtgcctggc ctcgcgccgc cgtgtatcgc
     1321 cccgccctgg gcggcaaggc tggcccggtc ggcaccagtt gcgtgagcgg aaagatggcc
     1381 gcttcccggc cctgctgcag ggagctcaaa atggaggacg cggcgctcgg gagagcgggc
     1441 gggtgagtca cccacacaaa ggaaaagggc ctttccgtcc tcagccgtcg cttcatgtga
     1501 ctccacggag taccgggcgc cgtccaggca cctcgattag ttctcgagct tttggagtac
     1561 gtcgtcttta ggttgggggg aggggtttta tgcgatggag tttccccaca ctgagtgggt
     1621 ggagactgaa gttaggccag cttggcactt gatgtaattc tccttggaat ttgccctttt
     1681 tgagtttgga tcttggttca ttctcaagcc tcagacagtg gttcaaagtt tttttcttcc
     1741 atttcaggtg tcgtgaggaa ttagcggtgg cggccatcac aagtttgtac aaaaaagcag
     1801 gctggcgccg gaaccaattc agtcgacggt accgcgggcc cgggatccac cggtcgccac
     1861 catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga
     1921 cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta
     1981 cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac
     2041 cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa
     2101 gcgccacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt
     2161 cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct
     2221 ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca
     2281 caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa
     2341 cggcatcaag gtgaacttca agacccgcca caacatcgag gacggcggtc tctgtcgagc
     2401 catgggcatt caagagagta tggccaggac agcttgtgaa tagttcagta actgtgcata
     2461 gttttatatt cattgtgagg caccgtgtca ccggtttgct gatttacaga gtattttaat
     2521 tgctaactgt atgctaccaa aatttccagt attcgaaaat aattttgctt gaatgtagaa
     2581 aaagaaaaaa gccaagaaat gtatgtgaaa cgagagtcta agggagcttt acctcagtct
     2641 cagaaaacat gcattccttc cttcatttag gaagcatgta ctggggtcta ctgtcagctt
     2701 gctattgtgt caaggagtag gagaatacaa aaatattaga gaatatgaat cacatctatt
     2761 aggagagttt tctacatacg cacattattc tgtcagtgac ataaggattt gagtcattca
     2821 gatttaaata cggtaggtac ctcaagttct cagatattat ttcattttct aaggttcgta
     2881 tttagttaat atgttatttt aatggcctta caaattctag attatctttt ttaaaaagtt
     2941 aaatagaacg taattgccat ttttatttaa tggtaaaaag catttttgtt tttgtgtgta
     3001 cttggttgta atattctcct tttcaattga gctatttttc tgatacttta ctcttaaaat
     3061 ttcattcagg aaaaaagtaa acaatattta agcttgacaa tcataaaaat gctctggtga
     3121 ctatagatta ttttaaaatt tattactgta gcttagggat atcttgatgg gatgctcctg
     3181 aaagcaatta attctcagtt ttttgtggct tctaatgcaa aatacattga cgcagacaga
     3241 atttgaaatg aattttcttc taatatagca attaatttta tttaaatatc tctagagttt
     3301 ttttttaata ctgtgactaa cctatgtttg ttcgctaccg tgagacctag acctacggcg
     3361 tgcagtgctt cagccgctac cccgaccaca tgaagcgcca cgacttcttc aagtccgcca
     3421 tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga
     3481 cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca
     3541 tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc
     3601 acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac ttcaagaccc
     3661 gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca
     3721 tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag tccgccctga
     3781 gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg
     3841 ggatcactca cggcatggac gagctgtaca agtaaagcgg ccgcactcga gatatctaga
     3901 cccagctttc ttgtacaaag tggtgatcta gctaaaacgc ggcctcgaat cgaattccgc
     3961 ccccccccct ctccctcccc cccccctaac gttactggcc gaagccgctt ggaataaggc
     4021 cggtgtgcgt ttgtctatat gttattttcc accatattgc cgtcttttgg caatgtgagg
     4081 gcccggaaac ctggccctgt cttcttgacg agcattccta ggggtctttc ccctctcgcc
     4141 aaaggaatgc aaggtctgtt gaatgtcgtg aaggaagcag ttcctctgga agcttcttga
     4201 agacaaacaa cgtctgtagc gaccctttgc aggcagcgga accccccacc tggcgacagg
     4261 tgcctctgcg gccaaaagcc acgtgtataa gatacacctg caaaggcggc acaaccccag
     4321 tgccacgttg tgagttggat agttgtggaa agagtcaaat ggctctcctc aagcgtattc
     4381 aacaaggggc tgaaggatgc ccagaaggta ccccattgta tgggatctga tctggggcct
     4441 cggtgcacat gctttacatg tgtttagtcg aggttaaaaa aacgtctagg ccccccgaac
     4501 cacggggacg tggttttcct ttgaaaaaca cgatgataat atggccacaa ccatgaccga
     4561 gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc cccagggccg tacgcaccct
     4621 cgccgccgcg ttcgccgact accccgccac gcgccacacc gtcgatccgg accgccacat
     4681 cgagcgggtc accgagctgc aagaactctt cctcacgcgc gtcgggctcg acatcggcaa
     4741 ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg accacgccgg agagcgtcga
     4801 agcgggggcg gtgttcgccg agatcggccc gcgcatggcc gagttgagcg gttcccggct
     4861 ggccgcgcag caacagatgg aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg
     4921 gttcctggcc accgtcggcg tctcgcccga ccaccagggc aagggtctgg gcagcgccgt
     4981 cgtgctcccc ggagtggagg cggccgagcg cgccggggtg cccgccttcc tggagacctc
     5041 cgcgccccgc aacctcccct tctacgagcg gctcggcttc accgtcaccg ccgacgtcga
     5101 ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag cccggtgcct gacgcccgcc
     5161 ccacgacccg cagcgcccga ccgaaaggag cgcacgaccc catgcatcga ttcgaatgta
     5221 cagatctgga tcctaggctt aagtatacct cgagttaaat tcactcctca ggtgcaggct
     5281 gcctatcaga aggtggtggc tggtgtggcc aatgccctgg ctcacaaata ccactgagat
     5341 ctttttccct ctgccaaaaa ttatggggac atcatgaagc cccttgagca tctgacttct
     5401 ggctaataaa ggaaatttat tttcattgca atagtgtgtt ggaatttttt gtgtctctca
     5461 ctcggaagga catatgggag ggcaaatcat ttaaaacatc agaatgagta tttggtttag
     5521 agtttggcaa catatgccca tatgctggct gccatgaaca aaggttggct ataaagaggt
     5581 catcagtata tgaaacagcc ccctgctgtc cattccttat tccatagaaa agccttgact
     5641 tgaggttaga ttttttttat attttgtttt gtgttatttt tttctttaac atccctaaaa
     5701 ttttccttac atgttttact agccagattt ttcctcctct cctgactact cccagtcata
     5761 gctgtccctc ttctcttatg gagatccctc gacttaatta accaacaagc tcgtcatcgc
     5821 tttgcagaag agcagagagg atatgctcat cgtctaaaga actacccatt ttattatata
     5881 ttagtcacga tatctataac aagaaaatat atatataata agttatcacg taagtagaac
     5941 atgaaataac aatataatta tcgtatgagt taaatcttaa aagtcacgta aaagataatc
     6001 atgcgtcatt ttgactcacg cggtcgttat agttcaaaat cagtgacact taccgcattg
     6061 acaagcacgc ctcacgggag ctccaagcgg cgactgagat gtcctaaatg cacagcgacg
     6121 gattcgcgct atttagaaag agagagcaat atttcaagaa tgcatgcgtc aattttacgc
     6181 agactatctt tctagggtta atacgtataa tacatatgat tcagctgcat taatgaatcg
     6241 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     6301 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     6361 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     6421 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     6481 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     6541 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     6601 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     6661 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     6721 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     6781 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     6841 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     6901 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     6961 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     7021 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     7081 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     7141 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     7201 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     7261 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     7321 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     7381 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     7441 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     7501 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     7561 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     7621 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     7681 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     7741 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     7801 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     7861 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     7921 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     7981 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     8041 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     8101 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     8161 aaaataaaca aataggggtt ccgcgcacat ttcccc
//
