LOCUS       sequence-267860-        6885 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For expression of ribozyme-flanked sgRNA after the Gateway (GW) LR 
            cloning..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6885)
  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 6885)
  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-GW-A
FEATURES             Location/Qualifiers
     source          1..6885
                     /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..2215
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(2240..2245,2246..2263,2264..2270)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             2324..2983
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(2390..2409)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             3325..3630
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     3509..3528
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(3671..3795)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(3960..3979)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4025..4080
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4079..4098)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4442..4464)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4453..4469
                     /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(4477..4483,4484..4501,4502..4507))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4522..4543
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4660..4677)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4831..5419)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4911..4930)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5888..6265)
                     /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     6212..6231
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6450..6554)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        complement(6591..6787)
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      6605..6740
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6858..6876
                     /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 tgaacgagaa acgtaaaatg atataaatat caatatatta aattagattt
     2161 tgcataaaaa acagactaca taatactgta aaacacaaca tatccagtca ctatggcggc
     2221 cgcattaggc accccaggct ttacacttta tgcttccggc tcgtataatg tgtggatttt
     2281 gagttaggat ccgtcgagat tttcaggagc taaggaagct aaaatggaga aaaaaatcac
     2341 tggatatacc accgttgata tatcccaatg gcatcgtaaa gaacattttg aggcatttca
     2401 gtcagttgct caatgtacct ataaccagac cgttcagctg gatattacgg cctttttaaa
     2461 gaccgtaaag aaaaataagc acaagtttta tccggccttt attcacattc ttgcccgcct
     2521 gatgaatgct catccggaat tccgtatggc aatgaaagac ggtgagctgg tgatatggga
     2581 tagtgttcac ccttgttaca ccgttttcca tgagcaaact gaaacgtttt catcgctctg
     2641 gagtgaatac cacgacgatt tccggcagtt tctacacata tattcgcaag atgtggcgtg
     2701 ttacggtgaa aacctggcct atttccctaa agggtttatt gagaatatgt ttttcgtctc
     2761 agccaatccc tgggtgagtt tcaccagttt tgatttaaac gtggccaata tggacaactt
     2821 cttcgccccc gttttcacca tgggcaaata ttatacgcaa ggcgacaagg tgctgatgcc
     2881 gctggcgatt caggttcatc atgccgtttg tgatggcttc catgtcggca gaatgcttaa
     2941 tgaattacaa cagtactgcg atgagtggca gggcggggcg taaacgcgtg gatccggctt
     3001 actaaaagcc agataacagt atgcgtattt gcgcgctgat ttttgcggta taagaatata
     3061 tactgatatg tatacccgaa gtatgtcaaa aagaggtatg ctatgaagca gcgtattaca
     3121 gtgacagttg acagcgacag ctatcagttg ctcaaggcat atatgatgtc aatatctccg
     3181 gtctggtaag cacaaccatg cagaatgaag cccgtcgtct gcgtgccgaa cgctggaaag
     3241 cggaaaatca ggaagggatg gctgaggtcg cccggtttat tgaaatgaac ggctcttttg
     3301 ctgacgagaa caggggctgg tgaaatgcag tttaaggttt acacctataa aagagagagc
     3361 cgttatcgtc tgtttgtgga tgtacagagt gatattattg acacgcccgg gcgacggatg
     3421 gtgatccccc tggccagtgc acgtctgctg tcagataaag tctcccgtga actttacccg
     3481 gtggtgcata tcggggatga aagctggcgc atgatgacca ccgatatggc cagtgtgccg
     3541 gtctccgtta tcggggaaga agtggctgat ctcagccacc gcgaaaatga catcaaaaac
     3601 gccattaacc tgatgttctg gggaatataa atgtcaggct cccttataca cagccagtct
     3661 gcaggtcgac catagtgact ggatatgttg tgttttacag tattatgtag tctgtttttt
     3721 atgcaaaatc taatttaata tattgatatt tatatcattt tacgtttctc gttcagcttt
     3781 cttgtacaaa gtggtgatct agctaaaacg cggcctcgaa tcgaattcga ttcgaatgta
     3841 cagatctgga tcctaggctt aagtatacct cgagttaaat tcactcctca ggtgcaggct
     3901 gcctatcaga aggtggtggc tggtgtggcc aatgccctgg ctcacaaata ccactgagat
     3961 ctttttccct ctgccaaaaa ttatggggac atcatgaagc cccttgagca tctgacttct
     4021 ggctaataaa ggaaatttat tttcattgca atagtgtgtt ggaatttttt gtgtctctca
     4081 ctcggaagga catatgggag ggcaaatcat ttaaaacatc agaatgagta tttggtttag
     4141 agtttggcaa catatgccca tatgctggct gccatgaaca aaggttggct ataaagaggt
     4201 catcagtata tgaaacagcc ccctgctgtc cattccttat tccatagaaa agccttgact
     4261 tgaggttaga ttttttttat attttgtttt gtgttatttt tttctttaac atccctaaaa
     4321 ttttccttac atgttttact agccagattt ttcctcctct cctgactact cccagtcata
     4381 gctgtccctc ttctcttatg gagatcaatt ctagcttggc gtaatcatgg tcatagctgt
     4441 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     4501 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     4561 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     4621 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     4681 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     4741 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     4801 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     4861 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     4921 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     4981 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcaatgct cacgctgtag
     5041 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     5101 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     5161 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     5221 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt
     5281 tggtatctgc gctctgctga agccacttac cttcggaaaa agagttggta gctcttgatc
     5341 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     5401 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     5461 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     5521 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     5581 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     5641 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     5701 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     5761 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     5821 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     5881 tttgcgcaac gttgtgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     5941 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     6001 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     6061 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     6121 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     6181 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     6241 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     6301 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     6361 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     6421 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     6481 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     6541 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg ggactagctt tttgcaaaag
     6601 cctaggcctc caaaaaagcc tcctcactac ttctggaata gctcagaggc cgaggcggcc
     6661 tcggcctctg cataaataaa aaaaattagt cagccatggg gcggagaatg ggcggaactg
     6721 ggcggagtta ggggcgggat gggcggagtt aggggcggga ctatggttgc tgactaattg
     6781 agatgtagct tgcatgccga catggattat tgactagtcc ctaagaaacc attattatca
     6841 tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtc
//
