LOCUS       sequence-273452-        8316 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 8316)
  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 8316)
  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..8316
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             118..751
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    798..923
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1416..1649
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1834..1878
                     /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    2165..2282
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2411..3589
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2642..3580
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3537..3557
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             join(3684..3686,3687..4400)
                     /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(3684..3686,3687..4397)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(3726..3747)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3987..4006)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4334..4355
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    4433..5006
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4600..4617)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4827..4846
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             5039..5638
                     /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(5039..5058)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5535..5555
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             5841..6074
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(6225..6242)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6231..6455
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     regulatory      6498..6507
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        6551..6655
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6656..6724,6725..7516)
                     /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(6874..6893)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7687..8275
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8176..8195
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 acggaccgcc actgccaatt acctgtggtt tcatttactc taaacctgtg attcctctga
       61 attattttca ttttaaagaa attgtatttg ttaaatatgt actacaaact tagtagttgg
      121 aagggctaat tcactcccaa agaagacaag atatccttga tctgtggatc taccacacac
      181 aaggctactt ccctgattag cagaactaca caccagggcc aggggtcaga tatccactga
      241 cctttggatg gtgctacaag ctagtaccag ttgagccaga taaggtagaa gaggccaata
      301 aaggagagaa caccagcttg ttacaccctg tgagcctgca tgggatggat gacccggaga
      361 gagaagtgtt agagtggagg tttgacagcc gcctagcatt tcatcacgtg gcccgagagc
      421 tgcatccgga gtacttcaag aactgctgat atcgagcttg ctacaaggga ctttccgctg
      481 gggactttcc agggaggcgt ggcctgggcg ggactgggga gtggcgagcc ctcagatcct
      541 gcatataagc agctgctttt tgcctgtact gggtctctct ggttagacca gatctgagcc
      601 tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga
      661 gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga
      721 cccttttagt cagtgtggaa aatctctagc agtggcgccc gaacagggac ttgaaagcga
      781 aagggaaacc agaggagctc tctcgacgca ggactcggct tgctgaagcg cgcacggcaa
      841 gaggcgaggg gcggcgactg gtgagtacgc caaaaatttt gactagcgga ggctagaagg
      901 agagagatgg gtgcgagagc gtcagtatta agcgggggag aattagatcg cgatgggaaa
      961 aaattcggtt aaggccaggg ggaaagaaaa aatataaatt aaaacatata gtatgggcaa
     1021 gcagggagct agaacgattc gcagttaatc ctggcctgtt agaaacatca gaaggctgta
     1081 gacaaatact gggacagcta caaccatccc ttcagacagg atcagaagaa cttagatcat
     1141 tatataatac agtagcaacc ctctattgtg tgcatcaaag gatagagata aaagacacca
     1201 aggaagcttt agacaagata gaggaagagc aaaacaaaag taagaccacc gcacagcaag
     1261 cggccgctga tcttcagacc tggaggagga gatatgaggg acaattggag aagtgaatta
     1321 tataaatata aagtagtaaa aattgaacca ttaggagtag cacccaccaa ggcaaagaga
     1381 agagtggtgc agagagaaaa aagagcagtg ggaataggag ctttgttcct tgggttcttg
     1441 ggagcagcag gaagcactat gggcgcagcg tcaatgacgc tgacggtaca ggccagacaa
     1501 ttattgtctg gtatagtgca gcagcagaac aatttgctga gggctattga ggcgcaacag
     1561 catctgttgc aactcacagt ctggggcatc aagcagctcc aggcaagaat cctggctgtg
     1621 gaaagatacc taaaggatca acagctcctg gggatttggg gttgctctgg aaaactcatt
     1681 tgcaccactg ctgtgccttg gaatgctagt tggagtaata aatctctgga acagatttgg
     1741 aatcacacga cctggatgga gtgggacaga gaaattaaca attacacaag cttaatacac
     1801 tccttaattg aagaatcgca aaaccagcaa gaaaagaatg aacaagaatt attggaatta
     1861 gataaatggg caagtttgtg gaattggttt aacataacaa attggctgtg gtatataaaa
     1921 ttattcataa tgatagtagg aggcttggta ggtttaagaa tagtttttgc tgtactttct
     1981 atagtgaata gagttaggca gggatattca ccattatcgt ttcagaccca cctcccaacc
     2041 ccgaggggac ccgacaggcc cgaaggaata gaagaagaag gtggagagag agacagagac
     2101 agatccattc gattagtgaa cggatctcga cggtatcgcc acaaatggca gtattcatcc
     2161 acaattttaa aagaaagggg gggattgggg ggtacagtgc aggggaaaga atagtagaca
     2221 taatagcaac agacatacaa actaaagaat tacaaaaaca aattacaaaa attcaaaatt
     2281 ttcgggttta ttacagggac agcagagatc cactttggat cgataagctt tgcaaagatg
     2341 gataaagttt taaacagaga ggaatctttg cagctaatgg accttctagg tcttgaaagg
     2401 agtgggaatt ggctccggtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga
     2461 gaagttgggg ggaggggtcg gcaattgaac cggtgcctag agaaggtggc gcggggtaaa
     2521 ctgggaaagt gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta
     2581 tataagtgca gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg ccagaacaca
     2641 ggtaagtgcc gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg gcccttgcgt
     2701 gccttgaatt acttccacct ggctgcagta cgtgattctt gatcccgagc ttcgggttgg
     2761 aagtgggtgg gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt
     2821 tgaggcctgg cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg
     2881 tctcgctgct ttcgataagt ctctagccat ttaaaatttt tgatgacctg ctgcgacgct
     2941 ttttttctgg caagatagtc ttgtaaatgc gggccaagat ctgcacactg gtatttcggt
     3001 ttttggggcc gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg
     3061 gggcctgcga gcgcggccac cgagaatcgg acgggggtag tctcaagctg gccggcctgc
     3121 tctggtgcct ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg
     3181 gtcggcacca gttgcgtgag cggaaagatg gccgcttccc ggccctgctg cagggagctc
     3241 aaaatggagg acgcggcgct cgggagagcg ggcgggtgag tcacccacac aaaggaaaag
     3301 ggcctttccg tcctcagccg tcgcttcatg tgactccacg gagtaccggg cgccgtccag
     3361 gcacctcgat tagttctcga gcttttggag tacgtcgtct ttaggttggg gggaggggtt
     3421 ttatgcgatg gagtttcccc acactgagtg ggtggagact gaagttaggc cagcttggca
     3481 cttgatgtaa ttctccttgg aatttgccct ttttgagttt ggatcttggt tcattctcaa
     3541 gcctcagaca gtggttcaaa gtttttttct tccatttcag gtgtcgtgag gaattctgag
     3601 gccaccatgt tgaaagaatt cagaatcagt gggatgaagt acaagaacac cttcagaacc
     3661 ggaggcaaca gttgaatgct agcgtgagca agggcgagga gctgttcacc ggggtggtgc
     3721 ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg
     3781 gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc
     3841 tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc
     3901 gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg
     3961 tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga
     4021 agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg
     4081 acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca
     4141 tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg
     4201 acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg
     4261 tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa gaccccaacg
     4321 agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca
     4381 tggacgagct gtacaagtaa ggatccgcat gcatctaggg cggccaattc cgcccctctc
     4441 ccyccccccc ccctaacgtt actggccgaa gccgcttgga ataaggccgg tgtgcgtttg
     4501 tctatatgtt attttccacc atattgccgt cttttggcaa tgtgagggcc cggaaacctg
     4561 gccctgtctt cttgacgagc attcctaggg gtctttcccc tctcgccaaa ggaatgcaag
     4621 gtctgttgaa tgtcgtgaag gaagcagttc ctctggaagc ttcttgaaga caaacaacgt
     4681 ctgtagcgac cctttgcagg cagcggaacc ccccacctgg cgacaggtgc ctctgcggcc
     4741 aaaagccacg tgtataagat acacctgcaa aggcggcaca accccagtgc cacgttgtga
     4801 gttggatagt tgtggaaaga gtcaaatggc tctcctcaag cgtattcaac aaggggctga
     4861 aggatgccca gaaggtaccc cattgtatgg gatctgatct ggggcctcgg tgcacatgct
     4921 ttacatgtgt ttagtcgagg ttaaaaaaac gtctaggccc cccgaaccac ggggacgtgg
     4981 ttttcctttg aaaaacacga tgataagctt gccacaaccc acaaggagac gaccttccat
     5041 gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac gacgtccccc gggccgtacg
     5101 caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg acccggaccg
     5161 ccacatcgag cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat
     5221 cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca cgccggagag
     5281 cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc
     5341 ccggctggcc gcgcagcaac agatggaagg cctcctggcg ccgcaccggc ccaaggagcc
     5401 cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac cagggcaagg gtctgggcag
     5461 cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga
     5521 gacctccgcg ccccgcaacc tccccttcta cgagcggctc ggcttcaccg tcaccgccga
     5581 cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgata
     5641 ataggcggcc gctcgagacc tagaaaaaca tggagcaatc acaagtagca atacagcagc
     5701 taccaatgct gattgtgcct ggctagaagc acaagaggag gaggaggtgg gttttccagt
     5761 cacacctcag gtacctttaa gaccaatgac ttacaaggca gctgtagatc ttagccactt
     5821 tttaaaagaa aaggggggac tggaagggct aattcactcc caacgaagac aagatctgct
     5881 ttttgcttgt actgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta
     5941 actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg
     6001 tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg
     6061 gaaaatctct agcagtagta gttcatgtca tcttattatt cagtatttat aacttgcaaa
     6121 gaaatgaata tcagagagtg agaggccttg acattataat agatttagca ggaattgaac
     6181 taggagtgga gcacacaggc aaagttctag agctcgctga tcagcctcga ctgtgccttc
     6241 tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc
     6301 cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg
     6361 tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa
     6421 tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg
     6481 ggggcgcgcc cctcgaggcc gccatggtca tagctgtttg acgtcaggtg gcacttttcg
     6541 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     6601 gctcacgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     6661 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     6721 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     6781 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     6841 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     6901 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     6961 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     7021 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     7081 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     7141 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     7201 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     7261 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     7321 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     7381 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     7441 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     7501 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     7561 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     7621 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     7681 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     7741 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     7801 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     7861 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     7921 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     7981 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     8041 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     8101 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     8161 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     8221 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     8281 cagcaacgcg gcctttttac ggttctggcc ttttgc
//
