LOCUS       sequence-404741-        8269 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Single-strand annealing (SSA) vector for monitoring crRNA(DMDex55)'s
            activity. DMD intron 55 region (downstream of exon 55). Puromycin 
            selectable..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8269)
  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 8269)
  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-EF1-alpha-mRxxFP1(DMDex55_10)-iP-A
FEATURES             Location/Qualifiers
     source          1..8269
                     /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      1842..1851
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(1978..1996)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3858..3881
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     protein_bind    complement(3973..3997)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    join(4039..4613,4614..4616,4617..4625)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4206..4223)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4433..4452
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4626..5225
                     /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(4626..4645)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5122..5142
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(5413..5432)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    5478..5533
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(5532..5551)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     repeat_region   6236..6270
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(6363..6380)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6534..7122)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6614..6633)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7293..8084,8085..8153))
                     /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     7916..7935
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8154..8258)
                     /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 agtcgactgg atccaccggt cgccaccatg gcctcctccg
     1861 aggacgtcat caaggagttc atgcgcttca aggtgcgcat ggagggctcc gtgaacggcc
     1921 acgagttcga gatcgagggc gagggcgagg gccgccccta cgagggcacc cagaccgcca
     1981 agctgaaggt gaccaagggc ggccccctgc ccttcgcctg ggacatcctg tcccctcagt
     2041 tccagtacgg ctccaaggcc tacgtgaagc accccgccga catccccgac tacttgaagc
     2101 tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac ggcggcgtgg
     2161 tgaccgtgac ccaggactcc tccctgcagg acggcgagtt catctacaag gtgaagctgc
     2221 gcggcaccaa cttcccctcc gacggccccg taatgcagaa gaagaccatg ggctgggagg
     2281 cctccaccga gcggatgtac cccgaggacg gcgccctgaa gggcgagatc aagatgaggc
     2341 tgaagctgaa ggacggcggc cactacgacg ccgaggtcaa gaccacctac atggtagggt
     2401 ctctgtcgag cccaactata cataacaaaa ctccataata gttcaggaaa attatatcag
     2461 gtattgagaa taaactttaa gagcctcctt gtactacaga atatgattct caccaagact
     2521 tcatttaaac aaaactaccc cacgttctgt tttcactgtt tatgcatatg gaactttatg
     2581 gaaataattt ttgtttctag gaaaaacaat tgtctgaaaa ttgtttgatc attaactacg
     2641 acaaattcag ccatctcatt cttattttca cagatcatgt taggaagaaa gctacaaaat
     2701 gccagattaa catttcaaaa catatttgaa agtaatgaag ggaaactgat tcagttagca
     2761 acgctaaata aaacagacaa ttcatacaga tgctttccct gtaaaatatt aaataaacac
     2821 tcaaaaatgt caacttttaa aatttaataa accaatccct aagttatttc tctgagcaaa
     2881 gtttctcctt gaccgaagct ctggttttat atgcaaatgt tttcctggtc agagcatgta
     2941 ctttttctaa gacgagggtg ttaagtggaa ttataaatga tgtttccttc tccctctgcc
     3001 tctctcctcc ttgtccaaat accgaaatac atcaggctgt ataaaagcaa ctattttgtt
     3061 ttgtccctgg cttgtcagtt acaagtacaa atgctgagaa ttgttcaatt ggatccacaa
     3121 gagtgctaaa gcggaaatgc ctgacttact tgccattgtt tcatcagctc ttttactccc
     3181 ttggagtctt ctaggagcct ttccttacgg gtagcatcct gtaggacatt ggcagttgtt
     3241 tcagcttctg taagccaggc aagaaacttt tccaggtcca gggggaactg ttgcagtaat
     3301 ctatgagttt cttccaaagc agcctctcgc tcactcaccc tgcaaaggac caaatgttca
     3361 gatgcaatta ttaaatatca gaatggtgca cctagtgaac tccataaaaa gagaaagatg
     3421 gaggaactaa attgtaatat accaacaatg gggtgagttg ttgcgctacc gtgagaccta
     3481 ggtgaagcac cccgccgaca tccccgacta cttgaagctg tccttccccg agggcttcaa
     3541 gtgggagcgc gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc
     3601 cctgcaggac ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga
     3661 cggccccgta atgcagaaga agaccatggg ctgggaggcc tccaccgagc ggatgtaccc
     3721 cgaggacggc gccctgaagg gcgagatcaa gatgaggctg aagctgaagg acggcggcca
     3781 ctacgacgcc gaggtcaaga ccacctacat ggccaagaag cccgtgcagc tgcccggcgc
     3841 ctacaagacc gacatcaagc tggacatcac ctcccacaac gaggactaca ccatcgtgga
     3901 acagtacgag cgcgccgagg gccgccactc caccggcgcc taatacggcc gctcgataag
     3961 cttgatatct agacccagct ttcttgtaca aagtggtgat ctagctaaaa cgcggcctcg
     4021 aatcgaattc cgcccccccc cctctccctc ccccccccct aacgttactg gccgaagccg
     4081 cttggaataa ggccggtgtg cgtttgtcta tatgttattt tccaccatat tgccgtcttt
     4141 tggcaatgtg agggcccgga aacctggccc tgtcttcttg acgagcattc ctaggggtct
     4201 ttcccctctc gccaaaggaa tgcaaggtct gttgaatgtc gtgaaggaag cagttcctct
     4261 ggaagcttct tgaagacaaa caacgtctgt agcgaccctt tgcaggcagc ggaacccccc
     4321 acctggcgac aggtgcctct gcggccaaaa gccacgtgta taagatacac ctgcaaaggc
     4381 ggcacaaccc cagtgccacg ttgtgagttg gatagttgtg gaaagagtca aatggctctc
     4441 ctcaagcgta ttcaacaagg ggctgaagga tgcccagaag gtaccccatt gtatgggatc
     4501 tgatctgggg cctcggtgca catgctttac atgtgtttag tcgaggttaa aaaaacgtct
     4561 aggccccccg aaccacgggg acgtggtttt cctttgaaaa acacgatgat aatatggcca
     4621 caaccatgac cgagtacaag cccacggtgc gcctcgccac ccgcgacgac gtccccaggg
     4681 ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc cacgcgccac accgtcgatc
     4741 cggaccgcca catcgagcgg gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc
     4801 tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc ggtggcggtc tggaccacgc
     4861 cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga
     4921 gcggttcccg gctggccgcg cagcaacaga tggaaggcct cctggcgccg caccggccca
     4981 aggagcccgc gtggttcctg gccaccgtcg gcgtctcgcc cgaccaccag ggcaagggtc
     5041 tgggcagcgc cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg gtgcccgcct
     5101 tcctggagac ctccgcgccc cgcaacctcc ccttctacga gcggctcggc ttcaccgtca
     5161 ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg catgacccgc aagcccggtg
     5221 cctgacgccc gccccacgac ccgcagcgcc cgaccgaaag gagcgcacga ccccatgcat
     5281 cgattcgaat gtacagatct ggatcctagg cttaagtata cctcgagtta aattcactcc
     5341 tcaggtgcag gctgcctatc agaaggtggt ggctggtgtg gccaatgccc tggctcacaa
     5401 ataccactga gatctttttc cctctgccaa aaattatggg gacatcatga agccccttga
     5461 gcatctgact tctggctaat aaaggaaatt tattttcatt gcaatagtgt gttggaattt
     5521 tttgtgtctc tcactcggaa ggacatatgg gagggcaaat catttaaaac atcagaatga
     5581 gtatttggtt tagagtttgg caacatatgc ccatatgctg gctgccatga acaaaggttg
     5641 gctataaaga ggtcatcagt atatgaaaca gccccctgct gtccattcct tattccatag
     5701 aaaagccttg acttgaggtt agattttttt tatattttgt tttgtgttat ttttttcttt
     5761 aacatcccta aaattttcct tacatgtttt actagccaga tttttcctcc tctcctgact
     5821 actcccagtc atagctgtcc ctcttctctt atggagatcc ctcgacttaa ttaaccaaca
     5881 agctcgtcat cgctttgcag aagagcagag aggatatgct catcgtctaa agaactaccc
     5941 attttattat atattagtca cgatatctat aacaagaaaa tatatatata ataagttatc
     6001 acgtaagtag aacatgaaat aacaatataa ttatcgtatg agttaaatct taaaagtcac
     6061 gtaaaagata atcatgcgtc attttgactc acgcggtcgt tatagttcaa aatcagtgac
     6121 acttaccgca ttgacaagca cgcctcacgg gagctccaag cggcgactga gatgtcctaa
     6181 atgcacagcg acggattcgc gctatttaga aagagagagc aatatttcaa gaatgcatgc
     6241 gtcaatttta cgcagactat ctttctaggg ttaatacgta taatacatat gattcagctg
     6301 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     6361 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     6421 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     6481 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     6541 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     6601 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     6661 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     6721 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     6781 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     6841 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     6901 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     6961 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     7021 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     7081 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     7141 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     7201 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     7261 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     7321 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     7381 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     7441 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     7501 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     7561 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     7621 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     7681 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     7741 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     7801 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     7861 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     7921 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     7981 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     8041 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     8101 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     8161 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     8221 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttcccc
//
