LOCUS       sequence-267191-        8315 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expressing a GFP reporter cassette, whose GFP reading frame is 
            disrupted by insertion of partial DMD exon 53 sequences.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8315)
  AUTHORS   Lyu P, Yoo KW, Yadav MK, Atala A, Aartsma-Rus A, Putten MV, Duan D, 
            Lu B
  TITLE     Sensitive and reliable evaluation of single-cut sgRNAs to restore 
            dystrophin by a GFP-reporter assay.
  JOURNAL   PLoS One. 2020 Sep 24;15(9):e0239468. doi: 
            10.1371/journal.pone.0239468. eCollection 2020.
  PUBMED    32970732
REFERENCE   2  (bases 1 to 8315)
  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: "PLoS One.";
            date: "2020-09-24"; volume: "15(9):e0239468. doi"; pages: " 
            10.1371/journal.pone.0239468. eCollection 2020"
COMMENT     Sequence Label: pSin-EF2-DMD53-GFP
FEATURES             Location/Qualifiers
     source          1..8315
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             117..750
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    797..922
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1415..1648
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1833..1877
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    2164..2281
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2409..3587
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2640..3578
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3535..3555
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             join(3682..3684,3685..4398)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(3682..3684,3685..4395)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(3724..3745)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3985..4004)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4332..4353
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    join(4431..5005,5006..5008,5009..5017)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4598..4615)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4825..4844
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             5037..5636
                     /codon_start=1
                     /gene="pac from Streptomyces"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVECPKDRATWCMTRKPGA"
     primer_bind     complement(5037..5056)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5533..5553
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             5839..6072
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(6223..6240)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6229..6453
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     regulatory      6496..6505
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        6549..6653
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6654..6722,6723..7514)
                     /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     complement(6872..6891)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7685..8273
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8174..8193
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cggaccgcca ctgccaatta cctgtggttt catttactct aaacctgtga ttcctctgaa
       61 ttattttcat tttaaagaaa ttgtatttgt taaatatgta ctacaaactt agtagttgga
      121 agggctaatt cactcccaaa gaagacaaga tatccttgat ctgtggatct accacacaca
      181 aggctacttc cctgattagc agaactacac accagggcca ggggtcagat atccactgac
      241 ctttggatgg tgctacaagc tagtaccagt tgagccagat aaggtagaag aggccaataa
      301 aggagagaac accagcttgt tacaccctgt gagcctgcat gggatggatg acccggagag
      361 agaagtgtta gagtggaggt ttgacagccg cctagcattt catcacgtgg cccgagagct
      421 gcatccggag tacttcaaga actgctgata tcgagcttgc tacaagggac tttccgctgg
      481 ggactttcca gggaggcgtg gcctgggcgg gactggggag tggcgagccc tcagatcctg
      541 catataagca gctgcttttt gcctgtactg ggtctctctg gttagaccag atctgagcct
      601 gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc ttgccttgag
      661 tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga tccctcagac
      721 ccttttagtc agtgtggaaa atctctagca gtggcgcccg aacagggact tgaaagcgaa
      781 agggaaacca gaggagctct ctcgacgcag gactcggctt gctgaagcgc gcacggcaag
      841 aggcgagggg cggcgactgg tgagtacgcc aaaaattttg actagcggag gctagaagga
      901 gagagatggg tgcgagagcg tcagtattaa gcgggggaga attagatcgc gatgggaaaa
      961 aattcggtta aggccagggg gaaagaaaaa atataaatta aaacatatag tatgggcaag
     1021 cagggagcta gaacgattcg cagttaatcc tggcctgtta gaaacatcag aaggctgtag
     1081 acaaatactg ggacagctac aaccatccct tcagacagga tcagaagaac ttagatcatt
     1141 atataataca gtagcaaccc tctattgtgt gcatcaaagg atagagataa aagacaccaa
     1201 ggaagcttta gacaagatag aggaagagca aaacaaaagt aagaccaccg cacagcaagc
     1261 ggccgctgat cttcagacct ggaggaggag atatgaggga caattggaga agtgaattat
     1321 ataaatataa agtagtaaaa attgaaccat taggagtagc acccaccaag gcaaagagaa
     1381 gagtggtgca gagagaaaaa agagcagtgg gaataggagc tttgttcctt gggttcttgg
     1441 gagcagcagg aagcactatg ggcgcagcgt caatgacgct gacggtacag gccagacaat
     1501 tattgtctgg tatagtgcag cagcagaaca atttgctgag ggctattgag gcgcaacagc
     1561 atctgttgca actcacagtc tggggcatca agcagctcca ggcaagaatc ctggctgtgg
     1621 aaagatacct aaaggatcaa cagctcctgg ggatttgggg ttgctctgga aaactcattt
     1681 gcaccactgc tgtgccttgg aatgctagtt ggagtaataa atctctggaa cagatttgga
     1741 atcacacgac ctggatggag tgggacagag aaattaacaa ttacacaagc ttaatacact
     1801 ccttaattga agaatcgcaa aaccagcaag aaaagaatga acaagaatta ttggaattag
     1861 ataaatgggc aagtttgtgg aattggttta acataacaaa ttggctgtgg tatataaaat
     1921 tattcataat gatagtagga ggcttggtag gtttaagaat agtttttgct gtactttcta
     1981 tagtgaatag agttaggcag ggatattcac cattatcgtt tcagacccac ctcccaaccc
     2041 cgaggggacc cgacaggccc gaaggaatag aagaagaagg tggagagaga gacagagaca
     2101 gatccattcg attagtgaac ggatctcgac ggtatcgcca caaatggcag tattcatcca
     2161 caattttaaa agaaaggggg ggattggggg gtacagtgca ggggaaagaa tagtagacat
     2221 aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt
     2281 tcgggtttat tacagggaca gcagagatcc actttggtcg ataagctttg caaagatgga
     2341 taaagtttta aacagagagg aatctttgca gctaatggac cttctaggtc ttgaaaggag
     2401 tgggaattgg ctccggtgcc cgtcagtggg cagagcgcac atcgcccaca gtccccgaga
     2461 agttgggggg aggggtcggc aattgaaccg gtgcctagag aaggtggcgc ggggtaaact
     2521 gggaaagtga tgtcgtgtac tggctccgcc tttttcccga gggtggggga gaaccgtata
     2581 taagtgcagt agtcgccgtg aacgttcttt ttcgcaacgg gtttgccgcc agaacacagg
     2641 taagtgccgt gtgtggttcc cgcgggcctg gcctctttac gggttatggc ccttgcgtgc
     2701 cttgaattac ttccacctgg ctgcagtacg tgattcttga tcccgagctt cgggttggaa
     2761 gtgggtggga gagttcgagg ccttgcgctt aaggagcccc ttcgcctcgt gcttgagttg
     2821 aggcctggcc tgggcgctgg ggccgccgcg tgcgaatctg gtggcacctt cgcgcctgtc
     2881 tcgctgcttt cgataagtct ctagccattt aaaatttttg atgacctgct gcgacgcttt
     2941 ttttctggca agatagtctt gtaaatgcgg gccaagatct gcacactggt atttcggttt
     3001 ttggggccgc gggcggcgac ggggcccgtg cgtcccagcg cacatgttcg gcgaggcggg
     3061 gcctgcgagc gcggccaccg agaatcggac gggggtagtc tcaagctggc cggcctgctc
     3121 tggtgcctgg cctcgcgccg ccgtgtatcg ccccgccctg ggcggcaagg ctggcccggt
     3181 cggcaccagt tgcgtgagcg gaaagatggc cgcttcccgg ccctgctgca gggagctcaa
     3241 aatggaggac gcggcgctcg ggagagcggg cgggtgagtc acccacacaa aggaaaaggg
     3301 cctttccgtc ctcagccgtc gcttcatgtg actccacgga gtaccgggcg ccgtccaggc
     3361 acctcgatta gttctcgagc ttttggagta cgtcgtcttt aggttggggg gaggggtttt
     3421 atgcgatgga gtttccccac actgagtggg tggagactga agttaggcca gcttggcact
     3481 tgatgtaatt ctccttggaa tttgcccttt ttgagtttgg atcttggttc attctcaagc
     3541 ctcagacagt ggttcaaagt ttttttcttc catttcaggt gtcgtgagga attctgaggc
     3601 caccatgttg aaagaattca gaatcagtgg gatgaagtac aagaacacct tcagaaccgg
     3661 aggcaacagt tgaatgctag cgtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc
     3721 atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc
     3781 gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg
     3841 cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc
     3901 taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc
     3961 caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag
     4021 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac
     4081 ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg
     4141 gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac
     4201 ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg
     4261 ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag
     4321 aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg
     4381 gacgagctgt acaagtaagg atccgcatgc atctagggcg gccaattccg cccctctccc
     4441 tccccccccc ctaacgttac tggccgaagc cgcttggaat aaggccggtg tgcgtttgtc
     4501 tatatgtgat tttccaccat attgccgtct tttggcaatg tgagggcccg gaaacctggc
     4561 cctgtcttct tgacgagcat tcctaggggt ctttcccctc tcgccaaagg aatgcaaggt
     4621 ctgttgaatg tcgtgaagga agcagttcct ctggaagctt cttgaagaca aacaacgtct
     4681 gtagcgaccc tttgcaggca gcggaacccc ccacctggcg acaggtgcct ctgcggccaa
     4741 aagccacgtg tataagatac acctgcaaag gcggcacaac cccagtgcca cgttgtgagt
     4801 tggatagttg tggaaagagt caaatggctc tcctcaagcg tattcaacaa ggggctgaag
     4861 gatgcccaga aggtacccca ttgtatggga tctgatctgg ggcctcggtg cacatgcttt
     4921 acatgtgttt agtcgaggtt aaaaaaacgt ctaggccccc cgaaccacgg ggacgtggtt
     4981 ttcctttgaa aaacacgatg ataagcttgc cacaacccac aaggagacga ccttccatga
     5041 ccgagtacaa gcccacggtg cgcctcgcca cccgcgacga cgtcccccgg gccgtacgca
     5101 ccctcgccgc cgcgttcgcc gactaccccg ccacgcgcca caccgtcgac ccggaccgcc
     5161 acatcgagcg ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg
     5221 gcaaggtgtg ggtcgcggac gacggcgccg cggtggcggt ctggaccacg ccggagagcg
     5281 tcgaagcggg ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg agcggttccc
     5341 ggctggccgc gcagcaacag atggaaggcc tcctggcgcc gcaccggccc aaggagcccg
     5401 cgtggttcct ggccaccgtc ggcgtctcgc ccgaccacca gggcaagggt ctgggcagcg
     5461 ccgtcgtgct ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga
     5521 cctccgcgcc ccgcaacctc cccttctacg agcggctcgg cttcaccgtc accgccgacg
     5581 tcgagtgccc gaaggaccgc gcgacctggt gcatgacccg caagcccggt gcctgataat
     5641 aggcggccgc tcgagaccta gaaaaacatg gagcaatcac aagtagcaat acagcagcta
     5701 ccaatgctga ttgtgcctgg ctagaagcac aagaggagga ggaggtgggt tttccagtca
     5761 cacctcaggt acctttaaga ccaatgactt acaaggcagc tgtagatctt agccactttt
     5821 taaaagaaaa ggggggactg gaagggctaa ttcactccca acgaagacaa gatctgcttt
     5881 ttgcttgtac tgggtctctc tggttagacc agatctgagc ctgggagctc tctggctaac
     5941 tagggaaccc actgcttaag cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg
     6001 cccgtctgtt gtgtgactct ggtaactaga gatccctcag acccttttag tcagtgtgga
     6061 aaatctctag cagtagtagt tcatgtcatc ttattattca gtatttataa cttgcaaaga
     6121 aatgaatatc agagagtgag aggccttgac attataatag atttagcagg aattgaacta
     6181 ggagtggagc acacaggcaa agttctagag ctcgctgatc agcctcgact gtgccttcta
     6241 gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca
     6301 ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc
     6361 attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata
     6421 gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg
     6481 ggcgcgcccc tcgaggccgc catggtcata gctgtttgac gtcaggtggc acttttcggg
     6541 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     6601 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     6661 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     6721 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     6781 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     6841 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     6901 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     6961 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     7021 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     7081 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     7141 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     7201 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     7261 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     7321 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     7381 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     7441 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     7501 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     7561 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     7621 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     7681 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     7741 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     7801 gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc
     7861 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     7921 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     7981 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     8041 cgacctacac cgaactgaga tacctacagc gtgagcattg agaaagcgcc acgcttcccg
     8101 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     8161 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     8221 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     8281 gcaacgcggc ctttttacgg ttcctggcct tttgc
//
