LOCUS       sequence-404743-        7262 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cloning vector for SSA vector (EGxxFP), 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 7262)
  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 7262)
  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-iP-A
FEATURES             Location/Qualifiers
     source          1..7262
                     /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     2875..2896
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(2966..2990)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    join(3032..3606,3607..3609,3610..3618)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3199..3216)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3426..3445
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3619..4218
                     /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(3619..3638)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4115..4135
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(4406..4425)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4471..4526
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4525..4544)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     repeat_region   5229..5263
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(5356..5373)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5527..6115)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5607..5626)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6286..7077,7078..7146))
                     /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     6909..6928
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7147..7251)
                     /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 tctgtcagcg
     2401 ctaccgtgag acctagacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa
     2461 gcgccacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt
     2521 cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct
     2581 ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca
     2641 caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa
     2701 cggcatcaag gtgaacttca agacccgcca caacatcgag gacggcagcg tgcagctcgc
     2761 cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca
     2821 ctacctgagc acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt
     2881 cctgctggag ttcgtgaccg ccgccgggat cactcacggc atggacgagc tgtacaagta
     2941 aagcggccgc actcgagata tctagaccca gctttcttgt acaaagtggt gatctagcta
     3001 aaacgcggcc tcgaatcgaa ttccgccccc ccccctctcc ctcccccccc cctaacgtta
     3061 ctggccgaag ccgcttggaa taaggccggt gtgcgtttgt ctatatgtta ttttccacca
     3121 tattgccgtc ttttggcaat gtgagggccc ggaaacctgg ccctgtcttc ttgacgagca
     3181 ttcctagggg tctttcccct ctcgccaaag gaatgcaagg tctgttgaat gtcgtgaagg
     3241 aagcagttcc tctggaagct tcttgaagac aaacaacgtc tgtagcgacc ctttgcaggc
     3301 agcggaaccc cccacctggc gacaggtgcc tctgcggcca aaagccacgt gtataagata
     3361 cacctgcaaa ggcggcacaa ccccagtgcc acgttgtgag ttggatagtt gtggaaagag
     3421 tcaaatggct ctcctcaagc gtattcaaca aggggctgaa ggatgcccag aaggtacccc
     3481 attgtatggg atctgatctg gggcctcggt gcacatgctt tacatgtgtt tagtcgaggt
     3541 taaaaaaacg tctaggcccc ccgaaccacg gggacgtggt tttcctttga aaaacacgat
     3601 gataatatgg ccacaaccat gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac
     3661 gacgtcccca gggccgtacg caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc
     3721 cacaccgtcg atccggaccg ccacatcgag cgggtcaccg agctgcaaga actcttcctc
     3781 acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg
     3841 gtctggacca cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc
     3901 atggccgagt tgagcggttc ccggctggcc gcgcagcaac agatggaagg cctcctggcg
     3961 ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac
     4021 cagggcaagg gtctgggcag cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc
     4081 ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc tccccttcta cgagcggctc
     4141 ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc
     4201 cgcaagcccg gtgcctgacg cccgccccac gacccgcagc gcccgaccga aaggagcgca
     4261 cgaccccatg catcgattcg aatgtacaga tctggatcct aggcttaagt atacctcgag
     4321 ttaaattcac tcctcaggtg caggctgcct atcagaaggt ggtggctggt gtggccaatg
     4381 ccctggctca caaataccac tgagatcttt ttccctctgc caaaaattat ggggacatca
     4441 tgaagcccct tgagcatctg acttctggct aataaaggaa atttattttc attgcaatag
     4501 tgtgttggaa ttttttgtgt ctctcactcg gaaggacata tgggagggca aatcatttaa
     4561 aacatcagaa tgagtatttg gtttagagtt tggcaacata tgcccatatg ctggctgcca
     4621 tgaacaaagg ttggctataa agaggtcatc agtatatgaa acagccccct gctgtccatt
     4681 ccttattcca tagaaaagcc ttgacttgag gttagatttt ttttatattt tgttttgtgt
     4741 tatttttttc tttaacatcc ctaaaatttt ccttacatgt tttactagcc agatttttcc
     4801 tcctctcctg actactccca gtcatagctg tccctcttct cttatggaga tccctcgact
     4861 taattaacca acaagctcgt catcgctttg cagaagagca gagaggatat gctcatcgtc
     4921 taaagaacta cccattttat tatatattag tcacgatatc tataacaaga aaatatatat
     4981 ataataagtt atcacgtaag tagaacatga aataacaata taattatcgt atgagttaaa
     5041 tcttaaaagt cacgtaaaag ataatcatgc gtcattttga ctcacgcggt cgttatagtt
     5101 caaaatcagt gacacttacc gcattgacaa gcacgcctca cgggagctcc aagcggcgac
     5161 tgagatgtcc taaatgcaca gcgacggatt cgcgctattt agaaagagag agcaatattt
     5221 caagaatgca tgcgtcaatt ttacgcagac tatctttcta gggttaatac gtataataca
     5281 tatgattcag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     5341 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     5401 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     5461 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     5521 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     5581 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     5641 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     5701 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     5761 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     5821 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     5881 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     5941 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     6001 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     6061 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     6121 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     6181 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     6241 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     6301 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     6361 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     6421 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     6481 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     6541 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     6601 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     6661 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     6721 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     6781 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     6841 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     6901 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     6961 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     7021 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     7081 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     7141 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     7201 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     7261 cc
//
