LOCUS       sequence-367532-        7580 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  fluorescent reporter for adenine base editing of mdx4cv point 
            mutation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7580)
  AUTHORS   Xu L, Zhang C, Li H, Wang P, Gao Y, Mokadam NA, Ma J, Arnold WD, Han
            R
  TITLE     Efficient precise in vivo base editing in adult dystrophic mice.
  JOURNAL   Nat Commun. 2021 Jun 17;12(1):3719. doi: 10.1038/s41467-021-23996-y.
  PUBMED    34140489
REFERENCE   2  (bases 1 to 7580)
  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: "Nat 
            Commun."; date: "2021-06-17"; volume: "12(1):3719. doi"; pages: " 
            10.1038/s41467-021-23996-y"
COMMENT     Sequence Label: pLKO‐puro‐2A‐mdx4cv‐GFP
FEATURES             Location/Qualifiers
     source          1..7580
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    89..110
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(125..130,131..148,149..155)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    163..179
                     /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)."
     primer_bind     168..190
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     187..203
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     187..203
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     222..242
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        224..242
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        270..496
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             497..677
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    724..849
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1342..1575
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1760..1804
                     /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    2109..2226
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     enhancer        2329..2632
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2633..2836
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2787..2807
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             2869..3465
                     /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(2869..2888)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3365..3385
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             3475..3534
                     /codon_start=1
                     /product="2A peptide from equine rhinitis A virus 
                     polyprotein"
                     /label=E2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="QCTNYALLKLAGDVESNPGP"
     CDS             join(3580..3582,3583..4296)
                     /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(3580..3582,3583..4293)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(3622..3643)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3883..3902)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4230..4251
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     LTR             4368..4601
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4673..4794
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4710..4729)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4764..4783
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4834..4969
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4989..5008)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4990..5008)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5018..5035)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5018..5034)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5027..5049)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5176..5631
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5263..5282)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5473..5494
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5657..5761
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5762..5830,5831..6622)
                     /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(5980..5999)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6793..7381
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7282..7301
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7535..7552
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact
       61 ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc
      121 aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat
      181 ttcacacagg aaacagctat gaccatgatt acgccaagcg cgcaattaac cctcactaaa
      241 gggaacaaaa gctggagctg caagcttaat gtagtcttat gcaatactct tgtagtcttg
      301 caacatggta acgatgagtt agcaacatgc cttacaagga gagaaaaagc accgtgcatg
      361 ccgattggtg gaagtaaggt ggtacgatcg tgccttatta ggaaggcaac agacgggtct
      421 gacatggatt ggacgaacca ctgaattgcc gcattgcaga gatattgtat ttaagtgcct
      481 agctcgatac ataaacgggt ctctctggtt agaccagatc tgagcctggg agctctctgg
      541 ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt
      601 gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct tttagtcagt
      661 gtggaaaatc tctagcagtg gcgcccgaac agggacttga aagcgaaagg gaaaccagag
      721 gagctctctc gacgcaggac tcggcttgct gaagcgcgca cggcaagagg cgaggggcgg
      781 cgactggtga gtacgccaaa aattttgact agcggaggct agaaggagag agatgggtgc
      841 gagagcgtca gtattaagcg ggggagaatt agatcgcgat gggaaaaaat tcggttaagg
      901 ccagggggaa agaaaaaata taaattaaaa catatagtat gggcaagcag ggagctagaa
      961 cgattcgcag ttaatcctgg cctgttagaa acatcagaag gctgtagaca aatactggga
     1021 cagctacaac catcccttca gacaggatca gaagaactta gatcattata taatacagta
     1081 gcaaccctct attgtgtgca tcaaaggata gagataaaag acaccaagga agctttagac
     1141 aagatagagg aagagcaaaa caaaagtaag accaccgcac agcaagcggc cgctgatctt
     1201 cagacctgga ggaggagata tgagggacaa ttggagaagt gaattatata aatataaagt
     1261 agtaaaaatt gaaccattag gagtagcacc caccaaggca aagagaagag tggtgcagag
     1321 agaaaaaaga gcagtgggaa taggagcttt gttccttggg ttcttgggag cagcaggaag
     1381 cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc agacaattat tgtctggtat
     1441 agtgcagcag cagaacaatt tgctgagggc tattgaggcg caacagcatc tgttgcaact
     1501 cacagtctgg ggcatcaagc agctccaggc aagaatcctg gctgtggaaa gatacctaaa
     1561 ggatcaacag ctcctgggga tttggggttg ctctggaaaa ctcatttgca ccactgctgt
     1621 gccttggaat gctagttgga gtaataaatc tctggaacag atttggaatc acacgacctg
     1681 gatggagtgg gacagagaaa ttaacaatta cacaagctta atacactcct taattgaaga
     1741 atcgcaaaac cagcaagaaa agaatgaaca agaattattg gaattagata aatgggcaag
     1801 tttgtggaat tggtttaaca taacaaattg gctgtggtat ataaaattat tcataatgat
     1861 agtaggaggc ttggtaggtt taagaatagt ttttgctgta ctttctatag tgaatagagt
     1921 taggcaggga tattcaccat tatcgtttca gacccacctc ccaaccccga ggggacccga
     1981 caggcccgaa ggaatagaag aagaaggtgg agagagagac agagacagat ccattcgatt
     2041 agtgaacgga tctcgacggt atcgatcacg agactagcct cgagacaaat ggcagtattc
     2101 atccacaatt ttaaaagaaa aggggggatt ggggggtaca gtgcagggga aagaatagta
     2161 gacataatag caacagacat acaaactaaa gaattacaaa aacaaattac aaaaattcaa
     2221 aattttcggg tttattacag ggacagcaga gatccacttt ggccgcggta gttattaata
     2281 gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact
     2341 tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat
     2401 gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta
     2461 tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc
     2521 tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg
     2581 ggactttcct acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg
     2641 gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct
     2701 ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa
     2761 atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt
     2821 ctatataagc agagctggtt tagtgaaccg tcagatccgc tagccaccat gaccgagtac
     2881 aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg caccctcgcc
     2941 gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg atccggaccg ccacatcgag
     3001 cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg
     3061 tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca cgccggagag cgtcgaagcg
     3121 ggggcggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc ccggctggcc
     3181 gcgcagcaac agatggaagg cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc
     3241 ctggccaccg tcggcgtgtc gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg
     3301 ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg
     3361 ccccgcaacc tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg
     3421 cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg gtgccctgta caagcaatgt
     3481 actaactacg ctttgttgaa actcgctggc gatgttgaaa gtaaccccgg tcctgaattc
     3541 caagaacagc tgcagaacag gagataacag ttgggatccg tgagcaaggg cgaggagctg
     3601 ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc
     3661 agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc
     3721 tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc
     3781 gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc
     3841 atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag
     3901 acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc
     3961 atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc
     4021 cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc
     4081 cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc
     4141 atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg
     4201 agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc
     4261 gggatcactc tcggcatgga cgagctgtac aagtaaggta cctttaagac caatgactta
     4321 caaggcagct gtagatctta gccacttttt aaaagaaaag gggggactgg aagggctaat
     4381 tcactcccaa cgaagacaag atctgctttt tgcttgtact gggtctctct ggttagacca
     4441 gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag
     4501 cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag
     4561 atccctcaga cccttttagt cagtgtggaa aatctctagc agtagtagtt catgtcatct
     4621 tattattcag tatttataac ttgcaaagaa atgaatatca gagagtgaga ggaacttgtt
     4681 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
     4741 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt
     4801 ctggctctag ctatcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc
     4861 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc
     4921 tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct agggacgtac
     4981 ccaattcgcc ctatagtgag tcgtattacg cgcgctcact ggccgtcgtt ttacaacgtc
     5041 gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg
     5101 ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc
     5161 tgaatggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     5221 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     5281 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     5341 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     5401 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     5461 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     5521 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     5581 tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac
     5641 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     5701 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     5761 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     5821 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     5881 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     5941 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     6001 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     6061 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     6121 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     6181 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     6241 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     6301 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     6361 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     6421 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     6481 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     6541 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     6601 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     6661 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     6721 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     6781 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     6841 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     6901 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     6961 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     7021 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     7081 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     7141 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     7201 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     7261 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     7321 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     7381 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     7441 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     7501 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     7561 agagcgccca atacgcaaac
//
