LOCUS       sequence-275571-        6286 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 6286)
  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 6286)
  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..6286
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    432..549
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     protein_bind    831..855
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     ncRNA           911..947
                     /product="self-cleaving hammerhead ribozyme (Ferre-D'Amare 
                     and Scott, 2010)"
                     /label=hammerhead ribozyme
                     /ncRNA_class="hammerhead_ribozyme"
     ncRNA           1063..1130
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     primer_bind     1137..1153
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1137..1153
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     regulatory      1158..1167
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1164..1853
                     /codon_start=1
                     /product="Anemonia majano cyan fluorescent protein"
                     /label=AmCyan1
                     /note="mammalian codon-optimized"
                     /translation="MALSNKFIGDDMKMTYHMDGCVNGHYFTVKGEGSGKPYEGTQTST
                     FKVTMANGGPLAFSFDILSTVFMYGNRCFTAYPTSMPDYFKQAFPDGMSYERTFTYEDG
                     GVATASWEISLKGNCFEHKSTFHGVNFPADGPVMAKKTTGWDPSFEKMTVCDGILKGDV
                     TAFLMLQGGGNYRCQFHTSYKTKKPVTMPPNHVVEHRIARTDLDKGGNSVQLTEHAVAH
                     ITSVVPF"
     primer_bind     complement(1883..1899)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1884..1900)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     protein_bind    complement(1910..1934)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(2099..2118)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2164..2219
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2218..2237)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(2569..2585)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2569..2585)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2582..2604)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2593..2609
                     /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(2617..2623,2624..2641,2642..2647))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2662..2683
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2800..2817)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2971..3559)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3051..3070)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3730..4521,4522..4590))
                     /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     4353..4372
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4591..4695)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4746..4881
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4998..5016
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5054..5076)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5176..5195
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5389..5411
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5403..5420
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5404..5420
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     LTR             5810..6067
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
ORIGIN
        1 ggccaggggg aaagaaacaa tataaactaa aacatatagt atgggcaagc agggagctag
       61 aacgattcgc agttaatcct ggccttttag agacatcaga aggctgtaga caaatactgg
      121 gacagctaca accatccctt cagacaggat cagaagaact tagatcatta tataatacaa
      181 tagcagtcct ctattgtgtg catcaaagga tagatgtaaa agacaccaag gaagccttag
      241 ataagataga ggaagagcaa aacaaaagta agaaaaaggc acagcaagca gcagctgaca
      301 caggaaacaa cagccaggtc agccaaaatt accctatagt gcagaacctc caggggcaaa
      361 tggtacatca ggccatatca cctagaactt taaattaaga cagcagtaca aatggcagta
      421 ttcatccaca attttaaaag aaaagggggg attggggggt acagtgcagg ggaaagaata
      481 gtagacataa tagcaacaga catacaaact aaagaattac aaaaacaaat tacaaaaatt
      541 caaaattttc gggtttatta cagggacagc agagatccag tttggaaagg accagcaaag
      601 ctcctctgga aaggtgaagg ggcagtagta atacaagata atagtgacat aaaagtagtg
      661 ccaagaagaa aagcaaagat catcagggat tatggaaaac agatggcagg tgatgattgt
      721 gtggcaagta gacaggatga ggattaacac atggaaaaga ttagtaaaac accatagctc
      781 tagagcgatc ccgatcttca gacctggagg aggagatatg agggacaatt acaagtttgt
      841 acaaaaaagc aggctggcgc cggaaccaat tcagtcgact ggatccggta ccgaattcgc
      901 ggccataccc ctgatgagtc cgtgaggacg aaacgagtaa gctcgtcggg tatcttacag
      961 gaactccgtt ttagagctat gctggaaaca gcatagcaag ttaaaataag gctagtccgt
     1021 tatcaacttg aaaaagtggc accgagtcgg tgcttttttt ttggccggca tggtcccagc
     1081 ctcctcgctg gcgccggctg ggcaacatgc ttcggcatgg cgaatgggac ggccgctcta
     1141 gaactagtgg atcccccgcc accatggccc tgtccaacaa gttcatcggc gacgacatga
     1201 agatgaccta ccacatggac ggctgcgtga acggccacta cttcaccgtg aagggcgagg
     1261 gcagcggcaa gccctacgag ggcacccaga cctccacctt caaggtgacc atggccaacg
     1321 gcggccccct ggccttctcc ttcgacatcc tgtccaccgt gttcatgtac ggcaaccgct
     1381 gcttcaccgc ctaccccacc agcatgcccg actacttcaa gcaggccttc cccgacggca
     1441 tgtcctacga gagaaccttc acctacgagg acggcggcgt ggccaccgcc agctgggaga
     1501 tcagcctgaa gggcaactgc ttcgagcaca agtccacctt ccacggcgtg aacttccccg
     1561 ccgacggccc cgtgatggcc aagaagacca ccggctggga cccctccttc gagaagatga
     1621 ccgtgtgcga cggcatcttg aagggcgacg tgaccgcctt cctgatgctg cagggcggcg
     1681 gcaactacag atgccagttc cacacctcct acaagaccaa gaagcccgtg accatgcccc
     1741 ccaaccacgt ggtggagcac cgcatcgcca gaaccgacct ggacaagggc ggcaacagcg
     1801 tgcagctgac cgagcacgcc gtggcccaca tcacctccgt ggtgcccttc tgagggctgc
     1861 aggaattcga tatcaagctt atcgataccg tcgacctcga gatatctaga cccagctttc
     1921 ttgtacaaag tggtgatcta gctaaaacgc ggcctcgaat cgaattcgat tcgaatgtac
     1981 agatctggat cctaggctta agtatacctc gagttaaatt cactcctcag gtgcaggctg
     2041 cctatcagaa ggtggtggct ggtgtggcca atgccctggc tcacaaatac cactgagatc
     2101 tttttccctc tgccaaaaat tatggggaca tcatgaagcc ccttgagcat ctgacttctg
     2161 gctaataaag gaaatttatt ttcattgcaa tagtgtgttg gaattttttg tgtctctcac
     2221 tcggaaggac atatgggagg gcaaatcatt taaaacatca gaatgagtat ttggtttaga
     2281 gtttggcaac atatgcccat atgctggctg ccatgaacaa aggttggcta taaagaggtc
     2341 atcagtatat gaaacagccc cctgctgtcc attccttatt ccatagaaaa gccttgactt
     2401 gaggttagat tttttttata ttttgttttg tgttattttt ttctttaaca tccctaaaat
     2461 tttccttaca tgttttacta gccagatttt tcctcctctc ctgactactc ccagtcatag
     2521 ctgtccctct tctcttatgg agatcaattc tagcttggcg taatcatggt catagctgtt
     2581 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     2641 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     2701 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     2761 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     2821 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     2881 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     2941 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     3001 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     3061 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     3121 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     3181 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     3241 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     3301 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     3361 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     3421 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     3481 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     3541 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     3601 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     3661 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     3721 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     3781 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     3841 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     3901 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     3961 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     4021 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     4081 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     4141 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     4201 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     4261 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     4321 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     4381 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     4441 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     4501 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     4561 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     4621 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     4681 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gggactagct ttttgcaaaa
     4741 gcctaggcct ccaaaaaagc ctcctcacta cttctggaat agctcagagg ccgaggcggc
     4801 ctcggcctct gcataaataa aaaaaattag tcagccatgg ggcggagaat gggcggaact
     4861 gggcggagtt aggggcggga tgggcggagt taggggcggg actatggttg ctgactaatt
     4921 gagatgagct tgcatgccga cattgattat tgactagtcc ctaagaaacc attcttatca
     4981 tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg
     5041 atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag
     5101 cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg
     5161 gctggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat atgcggtgtg
     5221 aaataccgca cagatgcgta aggagaaaat accgcatcag gcgccattcg ccattcaggc
     5281 tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga
     5341 aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac
     5401 gttgtaaaac gacggccagt gaattgcggc cgctggaagg gctaatttgg tcccaaaaaa
     5461 gacaagagat ccttgatctg tggatctacc acacacaagg ctacttccct gattggcaga
     5521 actacacacc agggccaggg atcagatatc cactgacctt tggatggtgc ttcaagttag
     5581 taccagttga accagagcaa gtagaagagg ccaatgaagg agagaacaac agcttgttac
     5641 accctatgag ccagcatggg atggaggacc cggagggaga agtattagtg tggaagtttg
     5701 acagcctcct agcatttcgt cacatggccc gagagctgca tccggagtac tacaaagact
     5761 gctgacatcg agctttctac aagggacttt ccgctgggga ctttccaggg aggtgtggcc
     5821 tgggcgggac tggggagtgg cgagccctca gatgctacat ataagcagct gctttttgcc
     5881 tgtactgggt ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg
     5941 aacccactgc ttaagcctca ataaagcttg ccttgagtgc tcaaagtagt gtgtgcccgt
     6001 ctgttgtgtg actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc
     6061 tctagcagtg gcgcccgaac agggacttga aagcgaaagt aaagccagag gagatctctc
     6121 gacgcaggac tcggcttgct gaagcgcgca cggcaagagg cgaggggcgg cgactggtga
     6181 gtacgccaaa aattttgact agcggaggct agaaggagag agatgggtgc gagagcgtcg
     6241 gtattaagcg ggggagaatt agataaatgg gaaaaaattc ggttaa
//
