LOCUS       sequence-97115-e        7250 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Knock-in donor vector to insert Exon 44 in front of Exon 45 of human
            DYSTROPHIN (DMD)gene, include EF1a-hygromycin resistance cassette 
            flanked by loxP sequences..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7250)
  AUTHORS   Li HL, Fujimoto N, Sasakawa N, Shirai S, Ohkame T, Sakuma T, Tanaka 
            M, Amano N, Watanabe A, Sakurai H, Yamamoto T, Yamanaka S, Hotta A
  TITLE     Precise Correction of the Dystrophin Gene in Duchenne Muscular 
            Dystrophy Patient Induced Pluripotent Stem Cells by TALEN and 
            CRISPR-Cas9.
  JOURNAL   Stem Cell Reports. 2014 Nov 25. pii: S2213-6711(14)00335-X. doi: 
            10.1016/j.stemcr.2014.10.013.
  PUBMED    25434822
REFERENCE   2  (bases 1 to 7250)
  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"; journalName: "Stem Cell 
            Reports. 2014 Nov 25. pii: S2213-6711(14)00335-X. doi: 
            10.1016/j.stemcr.2014.10.013."
COMMENT     Sequence Label: pENTR-DMD-Donor
FEATURES             Location/Qualifiers
     source          1..7250
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(63..651)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(143..162)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(741..1550)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     1464..1483
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1542..1561
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     protein_bind    1673..1772
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     protein_bind    2856..2889
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        3005..4187
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          3236..4178
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     4135..4155
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             4206..5231
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     protein_bind    5573..5606
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    complement(6794..6893)
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(6912..6935)
                     /label=pENTR-F
                     /note="pENTR vectors, forward primer"
     terminator      complement(6943..6970)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(7062..7148)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
       61 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
      121 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
      181 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
      241 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
      301 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
      361 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
      421 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
      481 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
      541 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
      601 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
      661 ttgatctttt ctacggggtc tgacgctcag tggaacgggg cccaatctga ataatgttac
      721 aaccaattaa ccaattctga ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta
      781 ttcatatcag gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa
      841 aactcaccga ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact
      901 cgtccaacat caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag
      961 aaatcaccat gagtgacgac tgaatccggt gagaatggca aaagtttatg catttctttc
     1021 cagacttgtt caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa
     1081 ccgttattca ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct gttaaaagga
     1141 caattacaaa caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata
     1201 ttttcacctg aatcaggata ttcttctaat acctggaatg ctgtttttcc ggggatcgca
     1261 gtggtgagta accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc
     1321 ataaattccg tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta
     1381 cctttgccat gtttcagaaa caactctggc gcatcgggct tcccatacaa gcgatagatt
     1441 gtcgcacctg attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc
     1501 atgttggaat ttaatcgcgg cctcgacgtt tcccgttgaa tatggctcat aacacccctt
     1561 gtattactgt ttatgtaagc agacagtttt attgttcatg atgatatatt tttatcttgt
     1621 gcaatgtaac atcagagatt ttgagacacg ggccagagct gcagctggat ggcaaataat
     1681 gattttattt tgactgatag tgacctgttc gttgcaacaa attgataagc aatgctttct
     1741 tataatgcca actttgtaca agaaagctgg gtctagatat ctcgagtgcg gccgcttgct
     1801 attgtgtcaa ggagtaggag aatacaaaaa tattagagaa tatgaatcac atctattagg
     1861 agagttttct acatacgcac attattctgt cagtgacata aggatttgag tcattcagat
     1921 ttaaatacgg taggtacctc aagttctcag atattatttc attttctaag gttcgtattt
     1981 agttaatatg ttattttaat ggccttacaa attctagatt atctttttta aaaagttaaa
     2041 tagaacgtaa ttgccatttt tatttaatgg taaaaagcat ttttgttttt gtgtgtactt
     2101 ggttgtaata ttctcctttt caattgagct atttttctga tactttactc ttaaaatttc
     2161 attcaggaaa aaagtaaaca atatttaagc ttgacaatca taaaaatgct ctggtgacta
     2221 tagattattt taaaatttat tactgtagct tagggatatc ttgatgggat gctcctgaaa
     2281 gcaattaatt ctcagttttt tgtggcttct aatgcaaaat acattgacgc agacagaatt
     2341 tgaaatgaat tttcttctaa tatagcaatt aattttattt aaatatctct agagtttttt
     2401 tttaatactg tgactaacct atgtttgttc tttttcacct ctcgtatcca cgatcactaa
     2461 gaaacccaaa tactttgttc atgtttaaat tttacaacat ttcatagact attaaacatg
     2521 gaacatcctt gtggggacaa gaaatcgaat ttgctcttga aaaggtttcc aactaattga
     2581 tttgtaggac attataacat cctctagctg acaagcttac aaaaataaaa actggagcta
     2641 accgagaggg tgcttttttc cctgacacat aaaaggtgtc tttctgtctt gtatcctttg
     2701 gatatgggca tgtcagtttc atagggaaat tttcacatgg agcttttgta tttctttctt
     2761 tgccagtaca actgcatgtg gtagcacact gtttaatctt ttctcaaata aaaagacatg
     2821 gggcttcatt tttgttttgc cggcgagctc agacgataac ttcgtataat gtatgctata
     2881 cgaagttatc aattgtctag acaattggcg gccctagaaa gtataggaac ttcattctac
     2941 cgggtagggg aggcgctttt cccaaggcag tctggagcgc taggtcttga aaggagtggg
     3001 aattggctcc ggtgcccgtc agtgggcaga gcgcacatcg cccacagtcc ccgagaagtt
     3061 ggggggaggg gtcggcaatt gaaccggtgc ctagagaagg tggcgcgggg taaactggga
     3121 aagtgatgtc gtgtactggc tccgcctttt tcccgagggt gggggagaac cgtatataag
     3181 tgcagtagtc gccgtgaacg ttctttttcg caacgggttt gccgccagaa cacaggtaag
     3241 tgccgtgtgt ggttcccgcg ggcctggcct ctttacgggt tatggccctt gcgtgccttg
     3301 aattacttcc acgcccctgg ctgcagtacg tgattcttga tcccgagctt cgggttggaa
     3361 gtgggtggga gagttcgagg ccttgcgctt aaggagcccc ttcgcctcgt gcttgagttg
     3421 aggcctggcc tgggcgctgg ggccgccgcg tgcgaatctg gtggcacctt cgcgcctgtc
     3481 tcgctgcttt cgataagtct ctagccattt aaaatttttg atgacctgct gcgacgcttt
     3541 ttttctggca agatagtctt gtaaatgcgg gccaagatct gcacactggt atttcggttt
     3601 ttggggccgc gggcggcgac ggggcccgtg cgtcccagcg cacatgttcg gcgaggcggg
     3661 gcctgcgagc gcggccaccg agaatcggac gggggtagtc tcaagctggc cggcctgctc
     3721 tggtgcctgg cctcgcgccg ccgtgtatcg ccccgccctg ggcggcaagg ctggcccggt
     3781 cggcaccagt tgcgtgagcg gaaagatggc cgcttcccgg ccctgctgca gggagctcaa
     3841 aatggaggac gcggcgctcg ggagagcggg cgggtgagtc acccacacaa aggaaaaggg
     3901 cctttccgtc ctcagccgtc gcttcatgtg actccacgga gtaccgggcg ccgtccaggc
     3961 acctcgatta gttctcgagc ttttggagta cgtcgtcttt aggttggggg gaggggtttt
     4021 atgcgatgga gtttccccac actgagtggg tggagactga agttaggcca gcttggcact
     4081 tgatgtaatt ctccttggaa tttgcccttt ttgagtttgg atcttggttc attctcaagc
     4141 ctcagacagt ggttcaaagt ttttttcttc catttcaggt gtcgtgagaa ttagcccggg
     4201 ccaccatgaa gaaacctgaa ctgacagcaa cttctgttga gaagtttctc attgaaaaat
     4261 ttgattctgt ttctgatctc atgcagctgt ctgaaggtga agaaagcaga gccttttctt
     4321 ttgatgttgg aggaagaggt tatgttctga gggtcaattc ttgtgctgat ggtttttaca
     4381 aagacagata tgtttacaga cactttgcct ctgctgctct gccaattcca gaagttctgg
     4441 acattggaga attttctgaa tctctcacct actgcatcag cagaagagca caaggagtca
     4501 ctctccagga tctccctgaa actgagctgc cagctgttct gcaacctgtt gctgaagcaa
     4561 tggatgccat tgcagcagct gatctgagcc aaacctctgg atttggtcct tttggtcccc
     4621 aaggcattgg tcagtacacc acttggaggg atttcatttg tgccattgct gatcctcatg
     4681 tctatcactg gcagactgtg atggatgaca cagtttctgc ttctgttgct caggcactgg
     4741 atgaactcat gctgtgggca gaagattgtc ctgaagtcag acacctggtc catgctgatt
     4801 ttggaagcaa caatgttctg acagacaatg gcagaatcac tgcagtcatt gactggtctg
     4861 aagccatgtt tggagattct caatatgagg ttgccaacat ttttttttgg agaccttggc
     4921 tggcttgcat ggaacaacaa acaagatatt ttgaaagaag acacccagaa ctggctggtt
     4981 cccccagact gagagcctac atgctcagaa ttggcctgga ccaactgtat caatctctgg
     5041 ttgatggaaa ctttgatgat gctgcttggg cacaaggaag atgtgatgcc attgtgaggt
     5101 ctggtgctgg aactgttgga agaactcaaa ttgcaagaag gtctgctgct gtttggactg
     5161 atggatgtgt tgaagttctg gctgactctg gaaacaggag accctccaca agacccagag
     5221 ccaaggaatg aatattagct agattatccc taatacctgc caccccactc ttaatcagtg
     5281 gtggaagaac ggtctcagaa ctgtttgttt caattggcca tttaagttta gtagtaaaag
     5341 actggttaat gataacaatg catcgtaaaa ccttcagaag gaaaggagaa tgttttgtgg
     5401 accactttgg ttttcttttt tgcgtgtggc agttttaagt tattagtttt taaaatcagt
     5461 actttttaat ggaaacaact tgaccaaaaa tttgtcacag aattttgaga cccattaaaa
     5521 aagttaaatg agaaacctgt gtgttccttt ggtcaacacc gagacaccta ggataacttc
     5581 gtataatgta tgctatacga agttatcact aattgcgttg cgcgatgttt tacataatcc
     5641 atctattttt cttgatccat atgcttttac ctgcaggcga tttgacagat ctgttgagaa
     5701 atggcggcgt tttcattatg atataaagat atttaatcag tggctaacag aagctgaaca
     5761 gtttctcaga aagacacaaa ttcctgagaa ttgggaacat gctaaataca aatggtatct
     5821 taaggaactc caggatggca ttgggcagcg ccagaccgtc gtgagaacat tgaatgcaac
     5881 tggggaagaa ataattcagc aatcctcaaa aacagatgcc agtattctac aggaaaaatt
     5941 gggaagcctg aatctgcggt ggcaggaggt ctgcaaacag ctgtcagaca gaaaaaagag
     6001 gtagggcgac agatctaata ggaatgaaaa cattttagca gactttttaa gctttcttta
     6061 gaagaatatt tcatgagaga ttataagcag ggtgaaaggc actaacatta aagaacctat
     6121 caaccattaa tcaacagcag taaagaaatt ttttatttct ttttttcata tactaaaata
     6181 tatacttgtg gctagttagt ggttttctgc tattttaaac ttgaagtttg ctttaaaaat
     6241 cacccatgat tgcttaaagg tgaatatctt caatatattt taacttcaac aagctgaatc
     6301 tcagttgttt ttcaagaaga ttttagaaag caattataaa tgattgtttt gtaggaaaga
     6361 cagatctttg cttagtttta aaaatagcta tgaatatgac tatgaagcta aaaaaagtga
     6421 tagtgtcact tacctctagt ttcaccacat ttgtgaatac attcttgaag gggaacttga
     6481 gccaaagagg tacaagttta atggggaaaa caaaacctca aaaaggttac tgtcaaattc
     6541 aatcatcatt taaatttccc ttggaatgta ttgaaggcac agaaagccaa atgcgtgctg
     6601 ctgcagttgg aaagcctaga gagtttataa atgggatttt gtattatgct tccagttgtt
     6661 gatgttaatg tgtcttgttt cgtaaaggaa gacttggcct ttatttacca aatgagacta
     6721 ttgttatgaa caatgaaaac ttcgttcttt tgccaagctc ttggatccag tcgactgaat
     6781 tggttccggc gccagcctgc ttttttgtac aaagttggca ttataaaaaa gcattgctta
     6841 tcaatttgtt gcaacgaaca ggtcactatc agtcaaaata aaatcattat ttggggcccg
     6901 agcttaagta actaactaac aggaagagtt tgtagaaacg caaaaaggcc atccgtcagg
     6961 atggccttct gcttagtttg atgcctggca gtttatggcg ggcgtcctgc ccgccaccct
     7021 ccgggccgtt gcttcacaac gttcaaatcc gctcccggcg gatttgtcct actcaggaga
     7081 gcgttcaccg acaaacaaca gataaaacga aaggcccagt cttccgactg agcctttcgt
     7141 tttatttgat gcctggcagt tccctactct cgcttagtag ttagacgtcc ccgagatcca
     7201 tgctagcggt aatacggtta tccacagaat caggggataa cgcaggaaag
//
