LOCUS       sequence-420038-        7642 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Piggybac transposon plasmid for a DMD exon 23 skipping reporter 
            (mCherry-DMDEx23).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7642)
  AUTHORS   Lessl AL, Pohmerer J, Lin Y, Wilk U, Hohn M, Horterer E, Wagner E, 
            Lachelt U
  TITLE     mCherry on Top: A Positive Read-Out Cellular Platform for Screening 
            DMD Exon Skipping Xenopeptide-PMO Conjugates.
  JOURNAL   Bioconjug Chem. 2023 Dec 20;34(12):2263-2274. doi: 
            10.1021/acs.bioconjchem.3c00408. Epub 2023 Nov 22.
  PUBMED    37991502
REFERENCE   2  (bases 1 to 7642)
  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: 
            "10.1021/acs.bioconjchem.3c00408"; journalName: "Bioconjug Chem"; 
            date: "2023-12-20- 20"; volume: "34"; issue: "12"; pages: 
            "2263-2274"
COMMENT     Sequence Label: PB-mCherry-DMDEx23-eGFP
FEATURES             Location/Qualifiers
     source          1..7642
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      21..30
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1001..1020
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     regulatory      1312..1321
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1318..1320,1321..1323,1324..2037)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1318..1320,1321..1323,1324..2034)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1363..1384)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1624..1643)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1971..1992
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2124..2143)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2189..2244
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2243..2262)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     repeat_region   2915..2949
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(2972..2988)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2972..2988)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2985..3007)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2996..3012
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(3020..3026,3027..3044,3045..3050))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3065..3086
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        3145..3340
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3191..3326
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    3346..3480
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3383..3402)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3437..3456
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3548..3565)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3719..4307)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3799..3818)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4478..5269,5270..5338))
                     /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     5101..5120
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5339..5443)
                     /gene="bla"
                     /label=AmpR promoter
     repeat_region   5481..5543
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     protein_bind    5840..5873
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     enhancer        5969..6272
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          6550..7566
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     7490..7512
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     7574..7593
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
ORIGIN
        1 ccggggactc agatctcgag gccaccatgg tgagcaaggg cgaggaggat aacatggcca
       61 tcatcaagga gttcatgcgc ttcaaggtgc acatggaggg ctccgtgaac ggccacgagt
      121 tcgagatcga ggtaagggca ctgagcagaa gggaagaagc tccgggggct ctttgtaggg
      181 tcctccagtc aggactcaaa cccagtagtg tctggttcca ggcactgacc ttgtatgtct
      241 cctggcccaa atgcccactc agggtagggg tgtagggcag acaacgagtc ttttgtcatc
      301 tacaggctct gcaaagttct ttgaaagagc aataaaatgg cttcaactat ctgagtgaca
      361 ctgtgaagga gatggccaag aaagcacctt cagaaatatg ccagaaatat ctgtcagaat
      421 ttgaagagat tgaggggcac tggaagaaac tttcctccca gttggtggaa agctgccaaa
      481 agctagaaga acatatgaat aaacttcgaa aatttcaggt aagccgaggt ttggccttgg
      541 aagaagctcc gggggctctt tgtagggtcc tccagtcagg actcaaaccc agtagtgtct
      601 ggttccaggc actgaccttg tatgtctcct ggcccaaatg cccactcagg gtaggggtgt
      661 agggcagaca acgagtcttt tgtcatctac agggcgaggg cgagggccgc ccctacgagg
      721 gcacccagac cgccaagctg gaggtgacca agggtggccc cctgcccttc gcctgggaca
      781 tcctgtcccc tcagttcatg tacggctcca aggcctacgt gaagcacccc gccgacatcc
      841 ccgactactt gaagctgtcc ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg
      901 aggacggcgg cgtggtgacc gtgacccagg actcctccct gcaggacggc gagttcatct
      961 acaaggtgaa gctgcgcggc accaacttcc cctccgacgg ccccgtaatg cagaagaaga
     1021 ccatgggctg ggaggcctcc tccgagcgga tgtaccccga ggacggcgcc ctgaagggcg
     1081 agatcaagca gaggctgaag ctgaaggacg gcggccacta cgacgctgag gtcaagacca
     1141 cctacaaggc caagaagccc gtgcagctgc ccggcgccta caacgtcaac atcaagttgg
     1201 acatcacctc ccacaacgag gactacacca tcgtggaaca gtacgaacgc gccgagggcc
     1261 gccactccac cggcggcatg gacgagctgt acaagtaagg atccaccggt cgccaccatg
     1321 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc
     1381 gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc
     1441 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc
     1501 gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag
     1561 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc
     1621 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg
     1681 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag
     1741 ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc
     1801 atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac
     1861 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac
     1921 ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg
     1981 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtaaagc
     2041 ggccgcactc ctcaggtgca ggctgcctat cagaaggtgg tggctggtgt ggccaatgcc
     2101 ctggctcaca aataccactg agatcttttt ccctctgcca aaaattatgg ggacatcatg
     2161 aagccccttg agcatctgac ttctggctaa taaaggaaat ttattttcat tgcaatagtg
     2221 tgttggaatt ttttgtgtct ctcactcgga aggacatatg ggagggcaaa tcatttaaaa
     2281 catcagaatg agtatttggt ttagagtttg gcaacatatg cccatatgct ggctgccatg
     2341 aacaaaggtt ggctataaag aggtcatcag tatatgaaac agccccctgc tgtccattcc
     2401 ttattccata gaaaagcctt gacttgaggt tagatttttt ttatattttg ttttgtgtta
     2461 tttttttctt taacatccct aaaattttcc ttacatgttt tactagccag atttttcctc
     2521 ctctcctgac tactcccagt catagctgtc cctcttctct tatggagatc cctcgacctg
     2581 cagagctcca gcctacacaa tcgctcaaga cgtgtaatgc ttttattata tattagtcac
     2641 gatatctata acaagaaaat atatatataa taagttatca cgtaagtaga acatgaaata
     2701 acaatataat tatcgtatga gttaaatctt aaaagtcacg taaaagataa tcatgcgtca
     2761 ttttgactca cgcggtcgtt atagttcaaa atcagtgaca cttaccgcat tgacaagcac
     2821 gcctcacggg agctccaagc ggcgactgag atgtcctaaa tgcacagcga cggattcgcg
     2881 ctatttagaa agagagagca atatttcaag aatgcatgcg tcaattttac gcagactatc
     2941 tttctagggt taaaagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     3001 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     3061 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     3121 gaaacctgtc gtgccagcgg atccgcatct caataagtca gcaaccatag tcccgcccct
     3181 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg
     3241 actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa
     3301 gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctaactt gtttattgca
     3361 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     3421 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggatc
     3481 cgctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     3541 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     3601 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     3661 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     3721 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     3781 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     3841 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     3901 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     3961 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     4021 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     4081 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     4141 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     4201 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     4261 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     4321 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     4381 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     4441 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     4501 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     4561 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     4621 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     4681 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     4741 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     4801 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     4861 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     4921 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     4981 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     5041 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     5101 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     5161 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     5221 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     5281 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     5341 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     5401 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     5461 tgccacctgg gtcgacttaa ccctagaaag ataatcatat tgtgacgtac gttaaagata
     5521 atcatgcgta aaattgacgc atgtgtttta tcgatctgta tatcgaggtt tatttattaa
     5581 tttgaataga tattaagttt tattatattt acacttacat actaataata aattcaacaa
     5641 acaatttatt tatgtttatt tatttattaa aaaaaaacaa aaactcaaaa tttcttctat
     5701 aaagtaacaa aacttttaaa cattctctct tttacaaaaa taaacttatt ttgtacttta
     5761 aaaacagtca tgttgtatta taaaataagt aattagctta acttatacat aatagaaaca
     5821 aattatactt attaatcgca taacttcgta taatgtatgc tatacgaagt tatgcgatta
     5881 agggatctgt agggcgcagt agtccaacta gttattaata gtaatcaatt acggggtcat
     5941 tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg
     6001 gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa
     6061 cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact
     6121 tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta
     6181 aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt
     6241 acatctacgt attagtcatc gctattacca tggtcgaggt gagccccacg ttctgcttca
     6301 ctctccccat ctcccccccc tccccacccc caattttgta tttatttatt ttttaattat
     6361 tttgtgcagc gatgggggcg gggggggggg gggsgcgcgc crgggggggg gggggggggg
     6421 gggggggggg gsggggsgrg gcggagaggt gcggcggcag ccaatcagag cggcgcgctc
     6481 cgaaagtttc cttttatggc gaggcggcgg cggcggcggc cctataaaaa gcgaagcgcg
     6541 cggcgggcgg gagtcgctgc gcgctgcctt cgccccgtgc cccgctccgc cgccgcctcg
     6601 cgccgcccgc cccggctctg actgaccgcg ttactcccac aggtgagcgg gcgggacggc
     6661 ccttctcctc cgggctgtaa ttagcgcttg gtttaatgac ggcttgtttc ttttctgtgg
     6721 ctgcgtgaaa gccttgaggg gctccgggag ggccctttgt gcggggggag cggctcgggg
     6781 ggtgcgtgcg tgtgtgtgtg cgtggggagc gccgcgtgcg gctccgcgct gcccggcggc
     6841 tgtgagcgct gcgggcgcgg cgcggggctt tgtgcgctcc gcagtgtgcg cgaggggagc
     6901 gcggccgggg gcggtgcccc gcggtgcggg gggggctgcg aggggaacaa aggctgcgtg
     6961 cggggtgtgt gcgtgggggg gtgagcaggg ggtgtgggcg cgtcggtcgg gctgcaaccc
     7021 cccctgcacc cccctccccg agttgctgag cacggcccgg cttcgggtgc ggggctccgt
     7081 acggggcgtg gcgcggggct cgccgtgccg ggcggggggt ggcggcaggt gggggtgccg
     7141 ggcggggcgg ggccgcctcg ggccggggrg ggctcggggg aggggcgcgg cggcccccgg
     7201 agcgccggcg gctgtcgagg cgcggcgagc cgcagccatt gccttttatg gtaatcgtgc
     7261 gagagggcgc agggacttcc tttgtcccaa atctgtgcgg agccgaaatc tgggaggcgc
     7321 cgccgcaccc cctctagcgg gcgcggggcg aagcggtgcg gcgccggcag gaaggaaatg
     7381 ggcggggagg gccttcgtgc gtcgccgcgc cgccgtcccc ttctccctct ccagcctcgg
     7441 ggctgtccgc ggggggacgg ctgccttcgg gggggacggg gcagggcggg gttcggcttc
     7501 tggcgtgtga ccggcggctc tagagcctct gctaaccatg ttcatgcctt cttctttttc
     7561 ctacagctcc tgggcaacgt gctggttatt gtgctgtctc atcattttgg caaagaattc
     7621 tgcagtcgac ggtaccgcgg gc
//
