LOCUS       sequence-404739-        7215 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cloning vector for SSA vector (mRxxFP), the target sequence can be 
            inserted by In-Fusion reaction after AfeI digestion.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7215)
  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 7215)
  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-mRxxFP1(AfeI)-iP-A
FEATURES             Location/Qualifiers
     source          1..7215
                     /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     2804..2827
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     protein_bind    complement(2919..2943)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    join(2985..3559,3560..3562,3563..3571)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3152..3169)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3379..3398
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3572..4171
                     /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(3572..3591)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4068..4088
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(4359..4378)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4424..4479
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4478..4497)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     repeat_region   5182..5216
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(5309..5326)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5480..6068)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5560..5579)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6239..7030,7031..7099))
                     /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     6862..6881
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7100..7204)
                     /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 ctctgtcagc gctaccgtga gacctaggtg aagcaccccg ccgacatccc cgactacttg
     2461 aagctgtcct tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga ggacggcggc
     2521 gtggtgaccg tgacccagga ctcctccctg caggacggcg agttcatcta caaggtgaag
     2581 ctgcgcggca ccaacttccc ctccgacggc cccgtaatgc agaagaagac catgggctgg
     2641 gaggcctcca ccgagcggat gtaccccgag gacggcgccc tgaagggcga gatcaagatg
     2701 aggctgaagc tgaaggacgg cggccactac gacgccgagg tcaagaccac ctacatggcc
     2761 aagaagcccg tgcagctgcc cggcgcctac aagaccgaca tcaagctgga catcacctcc
     2821 cacaacgagg actacaccat cgtggaacag tacgagcgcg ccgagggccg ccactccacc
     2881 ggcgcctaat acggccgctc gataagcttg atatctagac ccagctttct tgtacaaagt
     2941 ggtgatctag ctaaaacgcg gcctcgaatc gaattccgcc cccccccctc tccctccccc
     3001 ccccctaacg ttactggccg aagccgcttg gaataaggcc ggtgtgcgtt tgtctatatg
     3061 ttattttcca ccatattgcc gtcttttggc aatgtgaggg cccggaaacc tggccctgtc
     3121 ttcttgacga gcattcctag gggtctttcc cctctcgcca aaggaatgca aggtctgttg
     3181 aatgtcgtga aggaagcagt tcctctggaa gcttcttgaa gacaaacaac gtctgtagcg
     3241 accctttgca ggcagcggaa ccccccacct ggcgacaggt gcctctgcgg ccaaaagcca
     3301 cgtgtataag atacacctgc aaaggcggca caaccccagt gccacgttgt gagttggata
     3361 gttgtggaaa gagtcaaatg gctctcctca agcgtattca acaaggggct gaaggatgcc
     3421 cagaaggtac cccattgtat gggatctgat ctggggcctc ggtgcacatg ctttacatgt
     3481 gtttagtcga ggttaaaaaa acgtctaggc cccccgaacc acggggacgt ggttttcctt
     3541 tgaaaaacac gatgataata tggccacaac catgaccgag tacaagccca cggtgcgcct
     3601 cgccacccgc gacgacgtcc ccagggccgt acgcaccctc gccgccgcgt tcgccgacta
     3661 ccccgccacg cgccacaccg tcgatccgga ccgccacatc gagcgggtca ccgagctgca
     3721 agaactcttc ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg cggacgacgg
     3781 cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga
     3841 gatcggcccg cgcatggccg agttgagcgg ttcccggctg gccgcgcagc aacagatgga
     3901 aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca ccgtcggcgt
     3961 ctcgcccgac caccagggca agggtctggg cagcgccgtc gtgctccccg gagtggaggc
     4021 ggccgagcgc gccggggtgc ccgccttcct ggagacctcc gcgccccgca acctcccctt
     4081 ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac
     4141 ctggtgcatg acccgcaagc ccggtgcctg acgcccgccc cacgacccgc agcgcccgac
     4201 cgaaaggagc gcacgacccc atgcatcgat tcgaatgtac agatctggat cctaggctta
     4261 agtatacctc gagttaaatt cactcctcag gtgcaggctg cctatcagaa ggtggtggct
     4321 ggtgtggcca atgccctggc tcacaaatac cactgagatc tttttccctc tgccaaaaat
     4381 tatggggaca tcatgaagcc ccttgagcat ctgacttctg gctaataaag gaaatttatt
     4441 ttcattgcaa tagtgtgttg gaattttttg tgtctctcac tcggaaggac atatgggagg
     4501 gcaaatcatt taaaacatca gaatgagtat ttggtttaga gtttggcaac atatgcccat
     4561 atgctggctg ccatgaacaa aggttggcta taaagaggtc atcagtatat gaaacagccc
     4621 cctgctgtcc attccttatt ccatagaaaa gccttgactt gaggttagat tttttttata
     4681 ttttgttttg tgttattttt ttctttaaca tccctaaaat tttccttaca tgttttacta
     4741 gccagatttt tcctcctctc ctgactactc ccagtcatag ctgtccctct tctcttatgg
     4801 agatccctcg acttaattaa ccaacaagct cgtcatcgct ttgcagaaga gcagagagga
     4861 tatgctcatc gtctaaagaa ctacccattt tattatatat tagtcacgat atctataaca
     4921 agaaaatata tatataataa gttatcacgt aagtagaaca tgaaataaca atataattat
     4981 cgtatgagtt aaatcttaaa agtcacgtaa aagataatca tgcgtcattt tgactcacgc
     5041 ggtcgttata gttcaaaatc agtgacactt accgcattga caagcacgcc tcacgggagc
     5101 tccaagcggc gactgagatg tcctaaatgc acagcgacgg attcgcgcta tttagaaaga
     5161 gagagcaata tttcaagaat gcatgcgtca attttacgca gactatcttt ctagggttaa
     5221 tacgtataat acatatgatt cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     5281 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     5341 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     5401 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     5461 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     5521 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     5581 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     5641 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     5701 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     5761 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     5821 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     5881 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     5941 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     6001 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     6061 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     6121 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     6181 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     6241 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     6301 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     6361 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     6421 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     6481 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     6541 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     6601 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     6661 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     6721 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     6781 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     6841 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     6901 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     6961 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     7021 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     7081 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     7141 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     7201 cgcgcacatt tcccc
//
