LOCUS       sequence-267858-        6284 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses sgRNA targeting human DMD (Dystrophin) for packaging into 
            NanoMEDIC particle..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6284)
  AUTHORS   Gee P, Lung MSY, Okuzaki Y, Sasakawa N, Iguchi T, Makita Y, Hozumi 
            H, Miura Y, Yang LF, Iwasaki M, Wang XH, Waller MA, Shirai N, Abe 
            YO, Fujita Y, Watanabe K, Kagita A, Iwabuchi KA, Yasuda M, Xu H, 
            Noda T, Komano J, Sakurai H, Inukai N, Hotta A
  TITLE     Extracellular nanovesicles for packaging of CRISPR-Cas9 protein and 
            sgRNA to induce therapeutic exon skipping.
  JOURNAL   Nat Commun. 2020 Mar 13;11(1):1334. doi: 10.1038/s41467-020-14957-y.
  PUBMED    32170079
REFERENCE   2  (bases 1 to 6284)
  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: "2020-03-13"; volume: "11(1):1334. doi"; pages: " 
            10.1038/s41467-020-14957-y"
COMMENT     Sequence Label: PL-5LTR-RGR(DMD​#​1​)-AmCyan-A
FEATURES             Location/Qualifiers
     source          1..6284
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     LTR             785..1042
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1693..1810
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     protein_bind    2091..2115
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     ncRNA           2171..2207
                     /product="self-cleaving hammerhead ribozyme (Ferre-D'Amare 
                     and Scott, 2010)"
                     /label=hammerhead ribozyme
                     /ncRNA_class="hammerhead_ribozyme"
     ncRNA           2323..2390
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     primer_bind     2397..2413
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2397..2413
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     regulatory      2418..2427
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2424..3113
                     /codon_start=1
                     /product="Anemonia majano cyan fluorescent protein"
                     /label=AmCyan1
                     /note="mammalian codon-optimized"
                     /translation="MALSNKFIGDDMKMTYHMDGCVNGHYFTVKGEGSGKPYEGTQTST
                     FKVTMANGGPLAFSFDILSTVFMYGNRCFTAYPTSMPDYFKQAFPDGMSYERTFTYEDG
                     GVATASWEISLKGNCFEHKSTFHGVNFPADGPVMAKKTTGWDPSFEKMTVCDGILKGDV
                     TAFLMLQGGGNYRCQFHTSYKTKKPVTMPPNHVVEHRIARTDLDKGGNSVQLTEHAVAH
                     ITSVVPF"
     primer_bind     complement(3143..3159)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3144..3160)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     protein_bind    complement(3170..3194)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(3359..3378)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3424..3479
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3478..3497)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3841..3863)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3852..3868
                     /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(3876..3882,3883..3900,3901..3906))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3921..3942
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4059..4076)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4230..4818)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4310..4329)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5287..5664)
                     /label=ISS
                     /note="immunostimulatory sequence from the AmpR gene; 
                     contains unmethylated CpG dinucleotides in the context of 
                     5'-AACGTT-3' (Sato et al., 1996)"
     primer_bind     5611..5630
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5849..5953)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        complement(5990..6186)
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      6004..6139
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6257..6275
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt gcggccgctg gaagggctaa
      421 tttggtccca aaaaagacaa gagatccttg atctgtggat ctaccacaca caaggctact
      481 tccctgattg gcagaactac acaccagggc cagggatcag atatccactg acctttggat
      541 ggtgcttcaa gttagtacca gttgaaccag agcaagtaga agaggccaaa taaggagaga
      601 agaacagctt gttacaccct atgagccagc atgggatgga ggacccggag ggagaagtat
      661 tagtgtggaa gtttgacagc ctcctagcat ttcgtcacat ggcccgagag ctgcatccgg
      721 agtactacaa agactgctga catcgagctt tctacaaggg actttccgct ggggactttc
      781 cagggaggtg tggcctgggc gggactgggg agtggcgagc cctcagatgc tacatataag
      841 cagctgcttt ttgcctgtac tgggtctctc tggttagacc agatctgagc ctgggagctc
      901 tctggctaac tagggaaccc actgcttaag cctcaataaa gcttgccttg agtgctcaaa
      961 gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga gatccctcag acccttttag
     1021 tcagtgtgga aaatctctag cagtggcgcc cgaacaggga cttgaaagcg aaagtaaagc
     1081 cagaggagat ctctcgacgc aggactcggc ttgctgaagc gcgcacggca agaggcgagg
     1141 ggcggcgact ggtgagtacg ccaaaaattt tgactagcgg aggctagaag gagagagatg
     1201 ggtgcgagag cgtcggtatt aagcggggga gaattagata aatgggaaaa aattcggtta
     1261 aggccagggg gaaagaaaca atataaacta aaacatatag tatgggcaag cagggagcta
     1321 gaacgattcg cagttaatcc tggcctttta gagacatcag aaggctgtag acaaatactg
     1381 ggacagctac aaccatccct tcagacagga tcagaagaac ttagatcatt atataataca
     1441 atagcagtcc tctattgtgt gcatcaaagg atagatgtaa aagacaccaa ggaagcctta
     1501 gataagatag aggaagagca aaacaaaagt aagaaaaagg cacagcaagc agcagctgac
     1561 acaggaaaca acagccaggt cagccaaaat taccctatag tgcagaacct ccaggggcaa
     1621 atggtacatc aggccatatc acctagaact ttaaattaag acagcagtac aaatggcagt
     1681 attcatccac aattttaaaa gaaaaggggg gattgggggg tacagtgcag gggaaagaat
     1741 agtagacata atagcaacag acatacaaac taaagaatta caaaaacaaa ttacaaaaat
     1801 tcaaaatttt cgggtttatt acagggacag cagagatcca gtttggaaag gaccagcaaa
     1861 gctcctctgg aaaggtgaag gggcagtagt aatacaagat aatagtgaca taaaagtagt
     1921 gccaagaaga aaagcaaaga tcatcaggga ttatggaaaa cagatggcag gtgatgattg
     1981 tgtggcaagt agacaggatg aggattaaca catggaaaag attagtaaaa caccatagct
     2041 ctagagcgat ccgatcttca gacctggagg aggcgatatg agggacaatt acaagtttgt
     2101 acaaaaaagc aggctggcgc cggaaccaat tcagtcgact ggatccggta ccgaattcgc
     2161 ggccataccc ctgatgagtc cgtgaggacg aaacgagtaa gctcgtcggg tatcttacag
     2221 gaactccgtt ttagagctat gctggaaaca gcatagcaag ttaaaataag gctagtccgt
     2281 tatcaacttg aaaaagtggc accgagtcgg tgcttttttt ttggccggca tggtcccagc
     2341 ctcctcgctg gcgccggctg ggcaacatgc ttcggcatgg cgaatgggac ggccgctcta
     2401 gaactagtgg atcccccgcc accatggccc tgtccaacaa gttcatcggc gacgacatga
     2461 agatgaccta ccacatggac ggctgcgtga acggccacta cttcaccgtg aagggcgagg
     2521 gcagcggcaa gccctacgag ggcacccaga cctccacctt caaggtgacc atggccaacg
     2581 gcggccccct ggccttctcc ttcgacatcc tgtccaccgt gttcatgtac ggcaaccgct
     2641 gcttcaccgc ctaccccacc agcatgcccg actacttcaa gcaggccttc cccgacggca
     2701 tgtcctacga gagaaccttc acctacgagg acggcggcgt ggccaccgcc agctgggaga
     2761 tcagcctgaa gggcaactgc ttcgagcaca agtccacctt ccacggcgtg aacttccccg
     2821 ccgacggccc cgtgatggcc aagaagacca ccggctggga cccctccttc gagaagatga
     2881 ccgtgtgcga cggcatcttg aagggcgacg tgaccgcctt cctgatgctg cagggcggcg
     2941 gcaactacag atgccagttc cacacctcct acaagaccaa gaagcccgtg accatgcccc
     3001 ccaaccacgt ggtggagcac cgcatcgcca gaaccgacct ggacaagggc ggcaacagcg
     3061 tgcagctgac cgagcacgcc gtggcccaca tcacctccgt ggtgcccttc tgagggctgc
     3121 aggaattcga tatcaagctt atcgataccg tcgacctcga gatatctaga cccagctttc
     3181 ttgtacaaag tggtgatcta gctaaaacgc ggcctcgaat cgaattcgat tcgaatgtac
     3241 agatctggat cctaggctta agtatacctc gagttaaatt cactcctcag gtgcaggctg
     3301 cctatcagaa ggtggtggct ggtgtggcca atgccctggc tcacaaatac cactgagatc
     3361 tttttccctc tgccaaaaat tatggggaca tcatgaagcc ccttgagcat ctgacttctg
     3421 gctaataaag gaaatttatt ttcattgcaa tagtgtgttg gaattttttg tgtctctcac
     3481 tcggaaggac atatgggagg gcaaatcatt taaaacatca gaatgagtat ttggtttaga
     3541 gtttggcaac atatgcccat atgctggctg ccatgaacaa aggttggcta taaagaggtc
     3601 atcagtatat gaaacagccc cctgctgtcc attccttatt ccatagaaaa gccttgactt
     3661 gaggttagat tttttttata ttttgttttg tgttattttt ttctttaaca tccctaaaat
     3721 tttccttaca tgttttacta gccagatttt tcctcctctc ctgactactc ccagtcatag
     3781 ctgtccctct tctcttatgg agatcaattc tagcttggcg taatcatggt catagctgtt
     3841 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     3901 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     3961 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     4021 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     4081 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     4141 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     4201 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     4261 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     4321 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     4381 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcaatgctc acgctgtagg
     4441 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     4501 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     4561 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     4621 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt
     4681 ggtatctgcg ctctgctgaa gccacttacc ttcggaaaaa gagttggtag ctcttgatcc
     4741 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     4801 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     4861 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     4921 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     4981 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     5041 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     5101 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     5161 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     5221 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     5281 ttgcgcaacg ttgtgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     5341 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     5401 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     5461 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     5521 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     5581 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     5641 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     5701 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     5761 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     5821 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     5881 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     5941 taggggttcc gcgcacattt ccccgaaaag tgccacctgg gactagcttt ttgcaaaagc
     6001 ctaggcctcc aaaaaagcct cctcactact tctggaatag ctcagaggcc gaggcggcct
     6061 cggcctctgc ataaataaaa aaaattagtc agccatgggg cggagaatgg gcggaactgg
     6121 gcggagttag gggcgggatg ggcggagtta ggggcgggac tatggttgct gactaattga
     6181 gatgtagctt gcatgccgac atggattatt gactagtccc taagaaacca ttattatcat
     6241 gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtc
//
