LOCUS       sequence-309001-        6410 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Homology arms and linker-mEGFP sequence for C-terminus tagging of 
            human DMD via Cas9-excisable CAGGS-mCherry selection cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 6410)
  TITLE     Allen Institute for Cell Sciences Plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6410)
  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: 
            "Unpublished"
COMMENT     Sequence Label: AICSDP-55:DMD-mEGFP
FEATURES             Location/Qualifiers
     source          1..6410
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     polyA_signal    1103..1224
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1140..1159)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1194..1213
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     enhancer        1239..1618
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1620..1897
                     /label=chicken beta-actin promoter
     intron          1899..2916
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2840..2862
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2924..2943
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     primer_bind     complement(3144..3162)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3412..3431
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             3722..3775
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             3776..4375
                     /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(3776..3795)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4272..4292
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    4383..4607
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     CDS             4670..4684
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
     CDS             join(4697..4699,4700..5413)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(4739..4760)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5000..5019)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5347..5368
                     /label=EGFP-C
                     /note="EGFP, forward primer"
ORIGIN
        1 taaaactatg ggacatagac gaaaaggcaa gccataaagt aagtctcaga ttatcttact
       61 ctaaatctga attttgctac gaagtaccca gggacattgt gctgttcttt ttctccgtgc
      121 tgggagattc gtgctaggtc gtgctgcatt attagtaaaa ggctattgtg atagttaacc
      181 ctcatttagg aggagctcta agatggattc acaaaatacc caaatgcata caatacatgt
      241 gggtcacctt ataattaggg tctagagtaa tttggaatgt aaagcaattt ttattctgag
      301 gtttcctaat tcaaatgtta atagtgcacc aaaaggtcaa ttcaagagtt tattattatt
      361 atttttcaac ccaagtaaaa gcagagagaa aatagccacc tccaccatag cctcagaagc
      421 aagccaacag cctgaaacag ctttgaaatg aaaagttggt gtggcggtga tggtggcagt
      481 gataatggtg acgatggttg ggtgctggtg atggtagtgg tagttgtgaa ggtggtgatg
      541 gtggtttgat tgatagtaaa aaaaatgttc gttaatacaa gtagagagta agtaatcaat
      601 caatcactca tagccaaggt ggaaaagatg tatcccatca tggaatattc ctgttctgat
      661 agaaatcttg tgcttatcta tggaattctt ttgatatata tttacattgg gaacctgaat
      721 gtagcttgac atttttccat gtaaacacca gtagcctgat ccaacattaa gctgatacta
      781 acaaacaacg tgtaatggct tcattaataa ggctttgctt cttcctggaa actggtgaaa
      841 aatcaaacct tgttgtgtac accctcgatg cagcttctgt gttgtcttca cccagaaatg
      901 gggaatgatt tcccaaatgg caaagaaaca gagtgatgct atctatctgc accttttgta
      961 aagtctgtct ttctttctct ttgttttcca ggacacaatg cttgaagccg attacaagga
     1021 tgatgatgat cccggagagg ttcccgacat accaataact tcgtatagtg taggatatac
     1081 gaagttatat tagtattccg ataacttgtt tattgcagct tataatggtt acaaataaag
     1141 caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt
     1201 gtccaaactc atcaatgtat cttatcatgt ctggtctcga cattgattat tgactagtta
     1261 ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac
     1321 ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc
     1381 aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt
     1441 ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac
     1501 gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac
     1561 cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt
     1621 cgaggtgagc cccacgttct gcttcactct ccccatctcc cccccctccc cacccccaat
     1681 tttgtattta tttatttttt aattattttg tgcagcgatg ggggcggggg gggggggggg
     1741 gcgcgcgcca ggcggggcgg ggcggggcga ggggcggggc ggggcgaggc ggagaggtgc
     1801 ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct tttatggcga ggcggcggcg
     1861 gcggcggccc tataaaaagc gaagcgcgcg gcgggcgggg agtcgctgcg acgctgcctt
     1921 cgccccgtgc cccgctccgc cgccgcctcg cgccgcccgc cccggctctg actgaccgcg
     1981 ttactcccac aggtgagcgg gcgggacggc ccttctcctc cgggctgtaa ttagcgcttg
     2041 gtttaatgac ggcttgtttc ttttctgtgg ctgcgtgaaa gccttgaggg gctccgggag
     2101 ggccctttgt gcggggggag cggctcgggg ggtgcgtgcg tgtgtgtgtg cgtggggagc
     2161 gccgcgtgcg gctccgcgct gcccggcggc tgtgagcgct gcgggcgcgg cgcggggctt
     2221 tgtgcgctcc gcagtgtgcg cgaggggagc gcggccgggg gcggtgcccc gcggtgcggg
     2281 gggggctgcg aggggaacaa aggctgcgtg cggggtgtgt gcgtgggggg gtgagcaggg
     2341 ggtgtgggcg cgtcggtcgg gctgcaaccc cccctgcacc cccctccccg agttgctgag
     2401 cacggcccgg cttcgggtgc ggggctccgt acggggcgtg gcgcggggct cgccgtgccg
     2461 ggcggggggt ggcggcaggt gggggtgccg ggcggggcgg ggccgcctcg ggccggggag
     2521 ggctcggggg aggggcgcgg cggcccccgg agcgccggcg gctgtcgagg cgcggcgagc
     2581 cgcagccatt gccttttatg gtaatcgtgc gagagggcgc agggacttcc tttgtcccaa
     2641 atctgtgcgg agccgaaatc tgggaggcgc cgccgcaccc cctctagcgg gcgcggggcg
     2701 aagcggtgcg gcgccggcag gaaggaaatg ggcggggagg gccttcgtgc gtcgccgcgc
     2761 cgccgtcccc ttctccctct ccagcctcgg ggctgtccgc ggggggacgg ctgccttcgg
     2821 gggggacggg gcagggcggg gttcggcttc tggcgtgtga ccggcggctc tagagcctct
     2881 gctaaccatg ttcatgcctt cttctttttc ctacagctcc tgggcaacgt gctggttatt
     2941 gtgctgtctc atcattttgg caaagaattc cgccaccggg gatccaccgg agcttaccat
     3001 ggtctccaaa ggagaagaag ataacatggc catcatcaag gagttcatgc gcttcaaggt
     3061 gcacatggag ggctccgtga acggccacga gttcgagatc gagggcgagg gcgagggccg
     3121 cccctacgag ggcacccaga ccgccaagct gaaggtgacc aagggtggcc ccctgccctt
     3181 cgcctgggac atcctgtccc ctcagttcat gtacggctcc aaggcctacg tgaagcaccc
     3241 cgccgacatc cccgactact tgaagctgtc cttccccgag ggcttcaagt gggagcgcgt
     3301 gatgaacttc gaggacggcg gcgtggtgac cgtgacccag gactcctccc tgcaggacgg
     3361 cgagttcatc tacaaggtga agctgcgcgg caccaacttc ccctccgacg gccccgtaat
     3421 gcagaagaag accatgggct gggaggcctc ctccgagcgg atgtaccccg aggacggcgc
     3481 cctgaagggc gagatcaagc agaggctgaa gctgaaggac ggcggccact acgacgctga
     3541 ggtcaagacc acctacaagg ccaagaagcc cgtgcagctg cccggcgcct acaacgtcaa
     3601 catcaagttg gacatcacct cccacaacga ggactacacc atcgtggaac agtacgaacg
     3661 cgccgagggc cgccactcca ccggcggaat ggatgaactc tataaagaat tcggcagtgg
     3721 agagggcaga ggaagtctgc taacatgcgg tgacgtcgag gagaatcctg gcccaatgac
     3781 cgagtacaag cccacggtgc gcctcgccac ccgcgacgac gtccccaggg ccgtacgcac
     3841 cctcgccgcc gcgttcgccg actaccccgc cacgcgccac accgtcgatc cggaccgcca
     3901 catcgagcgg gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg
     3961 caaggtgtgg gtcgcggacg acggcgccgc ggtggcggtc tggaccacgc cggagagcgt
     4021 cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga gcggttcccg
     4081 gctggccgcg cagcaacaga tggaaggcct cctggcgccg caccggccca aggagcccgc
     4141 gtggttcctg gccaccgtcg gagtctcgcc cgaccaccag ggcaagggtc tgggcagcgc
     4201 cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg gtgcccgcct tcctggagac
     4261 ctccgcgccc cgcaacctcc ccttctacga gcggctcggc ttcaccgtca ccgccgacgt
     4321 cgaggtgccc gaaggaccgc gcacctggtg catgacccgc aagcccggtg cctgaggtac
     4381 tcctgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
     4441 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg
     4501 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga
     4561 ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggaaa taacttcgta
     4621 tagtgtagga tatacgaagt tatataggta tgtcgggaac ctctccgggg atgatgatga
     4681 taagagatct gaattcgtga gcaagggcga ggagctgttc accggggtgg tgcccatcct
     4741 ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg
     4801 cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt
     4861 gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc
     4921 cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga
     4981 gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga
     5041 gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa
     5101 catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga
     5161 caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag
     5221 cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct
     5281 gcccgacaac cactacctga gcacccagtc caagctgagc aaagacccca acgagaagcg
     5341 cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga
     5401 gctgtacaag taggaagtct ttttcacatg gcagatgatt tgggcagagc gatggagtcc
     5461 ttagtatcag tcatgacaga tgaagaagga gcagaataaa tgttttacaa ctcctgattc
     5521 ccgcatggtt tttataatat tcatacaaca aagaggatta gacagtaaga gtttacaaga
     5581 aataaatcta tatttttgtg aagggtagtg gtattatact gtagatttca gtagtttcta
     5641 agtctgttat tgttttgtta acaatggcag gttttacacg tctatgcaat tgtacaaaaa
     5701 agttataaga aaactacatg taaaatcttg atagctaaat aacttgccat ttctttatat
     5761 ggaacgcatt ttgggttgtt taaaaattta taacagttat aaagaaagat tgtaaactaa
     5821 agtgtgcttt ataaaaaaaa gttgtttata aaaaccccta aaaacaaaac aaacacacac
     5881 acacacacat acacacacac acacaaaact ttgaggcagc gcattgtttt gcatcctttt
     5941 ggcgtgatat ccatatgaaa ttcatggctt tttctttttt tgcatattaa agataagact
     6001 tcctctacca ccacaccaaa tgactactac acactgctca tttgagaact gtcagctgag
     6061 tggggcaggc ttgagttttc atttcatata tctatatgtc tataagtata taaatactat
     6121 agttatatag ataaagagat acgaatttct atagactgac tttttccatt ttttaaatgt
     6181 tcatgtcaca tcctaataga aagaaattac ttctagtcag tcatccaggc ttacctgctt
     6241 ggtctagaat ggatttttcc cggagccgga agccaggagg aaactacacc acactaaaac
     6301 attgtctaca gctccagatg tttctcattt taaacaactt tccactgaca acgaaagtaa
     6361 agtaaagtat tggatttttt taaagggaac atgtgaatga atacacagga
//
