LOCUS       sequence-275574-        6887 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 6887)
  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 6887)
  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..6887
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     11..30
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(join(150..156,157..174,175..180))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    complement(205..329)
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     misc_feature    611..728
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     LTR             1379..1636
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(2026..2043)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2026..2042)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2035..2057)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2251..2270)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2370..2392
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2430..2448)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     rep_origin      2565..2700
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        2751..2855
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2856..2924,2925..3716)
                     /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(3074..3093)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3887..4475
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4376..4395
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4629..4646
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4763..4784
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4799..4804,4805..4822,4823..4829)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4837..4853
                     /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     4842..4864
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4861..4877
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4861..4877
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     5209..5228
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     polyA_signal    5227..5282
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     5328..5347
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     protein_bind    5512..5636
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(5677..5982)
                     /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     complement(5779..5798)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(join(6324..6887,1..96))
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
ORIGIN
        1 ggtacattga gcaactgact gaaatgcctc aaaatgttct ttacgatgcc attgggatat
       61 atcaacggtg gtatatccag tgattttttt ctccatttta gcttccttag ctcctgaaaa
      121 tctcgacgga tcctaactca aaatccacac attatacgag ccggaagcat aaagtgtaaa
      181 gcctggggtg cctaatgcgg ccgccatagt gactggatat gttgtgtttt acagtattat
      241 gtagtctgtt ttttatgcaa aatctaattt aatatattga tatttatatc attttacgtt
      301 tctcgttcag cttttttgta caaacttgta attgtccctc atatctcctc ctccaggtct
      361 gaagatcggg atcgctctag agctatggtg ttttactaat cttttccatg tgttaatcct
      421 catcctgtct acttgccaca caatcatcac ctgccatctg ttttccataa tccctgatga
      481 tctttgcttt tcttcttggc actactttta tgtcactatt atcttgtatt actactgccc
      541 cttcaccttt ccagaggagc tttgctggtc ctttccaaac tggatctctg ctgtccctgt
      601 aataaacccg aaaattttga atttttgtaa tttgtttttg taattcttta gtttgtatgt
      661 ctgttgctat tatgtctact attctttccc ctgcactgta ccccccaatc cccccttttc
      721 ttttaaaatt gtggatgaat actgccattt gtactgctgt cttaatttaa agttctaggt
      781 gatatggcct gatgtaccat ttgcccctgg aggttctgca ctatagggta attttggctg
      841 acctggctgt tgtttcctgt gtcagctgct gcttgctgtg cctttttctt acttttgttt
      901 tgctcttcct ctatcttatc taaggcttcc ttggtgtctt ttacatctat cctttgatgc
      961 acacaataga ggactgctat tgtattatat aatgatctaa gttcttctga tcctgtctga
     1021 agggatggtt gtagctgtcc cagtatttgt ctacagcctt ctgatgtctc taaaaggcca
     1081 ggattaactg cgaatcgttc tagctccctg cttgcccata ctatatgttt tagtttatat
     1141 tgtttctttc cccctggcct taaccgaatt ttttcccatt tatctaattc tcccccgctt
     1201 aataccgacg ctctcgcacc catctctctc cttctagcct ccgctagtca aaatttttgg
     1261 cgtactcacc agtcgccgcc cctcgcctct tgccgtgcgc gcttcagcaa gccgagtcct
     1321 gcgtcgagag atctcctctg gctttacttt cgctttcaag tccctgttcg ggcgccactg
     1381 ctagagattt tccacactga ctaaaagggt ctgagggatc tctagttacc agagtcacac
     1441 aacagacggg cacacactac tttgagcact caaggcaagc tttattgagg cttaagcagt
     1501 gggttcccta gttagccaga gagctcccag gctcagatct ggtctaacca gagagaccca
     1561 gtacaggcaa aaagcagctg cttatatgta gcatctgagg gctcgccact ccccagtccc
     1621 gcccaggcca cacctccctg gaaagtcccc agcggaaagt cccttgtaga aagctcgatg
     1681 tcagcagtct ttgtagtact ccggatgcag ctctcgggcc atgtgacgaa atgctaggag
     1741 gctgtcaaac ttccacacta atacttctcc ctccgggtcc tccatcccat gctggctcat
     1801 agggtgtaac aagctgttgt tctctccttc attggcctct tctacttgct ctggttcaac
     1861 tggtactaac ttgaagcacc atccaaaggt cagtggatat ctgatccctg gccctggtgt
     1921 gtagttctgc caatcaggga agtagccttg tgtgtggtag atccacagat caaggatctc
     1981 ttgtcttttt tgggaccaaa ttagcccttc cagcggccgc aattcactgg ccgtcgtttt
     2041 acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc
     2101 ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt
     2161 gcgcagcctg aatggcgaat ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg
     2221 tatttcacac cgcatatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     2281 ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     2341 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     2401 gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa
     2461 tgtcatgata agaatggttt cttagggact agtcaataat caatgtcggc atgcaagctc
     2521 atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc
     2581 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg
     2641 ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc
     2701 taggcttttg caaaaagcta gtcccaggtg gcacttttcg gggaaatgtg cgcggaaccc
     2761 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     2821 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     2881 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     2941 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     3001 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     3061 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac
     3121 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     3181 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     3241 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     3301 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     3361 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc
     3421 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     3481 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     3541 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc
     3601 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     3661 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     3721 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     3781 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     3841 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     3901 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     3961 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     4021 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     4081 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     4141 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     4201 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     4261 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     4321 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     4381 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     4441 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     4501 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     4561 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
     4621 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc
     4681 tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag
     4741 cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt
     4801 tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca
     4861 caggaaacag ctatgaccat gattacgcca agctagaatt gatctccata agagaagagg
     4921 gacagctatg actgggagta gtcaggagag gaggaaaaat ctggctagta aaacatgtaa
     4981 ggaaaatttt agggatgtta aagaaaaaaa taacacaaaa caaaatataa aaaaaatcta
     5041 acctcaagtc aaggcttttc tatggaataa ggaatggaca gcagggggct gtttcatata
     5101 ctgatgacct ctttatagcc aacctttgtt catggcagcc agcatatggg catatgttgc
     5161 caaactctaa accaaatact cattctgatg ttttaaatga tttgccctcc catatgtcct
     5221 tccgagtgag agacacaaaa aattccaaca cactattgca atgaaaataa atttccttta
     5281 ttagccagaa gtcagatgct caaggggctt catgatgtcc ccataatttt tggcagaggg
     5341 aaaaagatct cagtggtatt tgtgagccag ggcattggcc acaccagcca ccaccttctg
     5401 ataggcagcc tgcacctgag gagtgaattt aactcgaggt atacttaagc ctaggatcca
     5461 gatctgtaca ttcgaatcga attcgattcg aggccgcgtt ttagctagat caccactttg
     5521 tacaagaaag ctgaacgaga aacgtaaaat gatataaata tcaatatatt aaattagatt
     5581 ttgcataaaa aacagactac ataatactgt aaaacacaac atatccagtc actatggtcg
     5641 acctgcagac tggctgtgta taagggagcc tgacatttat attccccaga acatcaggtt
     5701 aatggcgttt ttgatgtcat tttcgcggtg gctgagatca gccacttctt ccccgataac
     5761 ggagaccggc acactggcca tatcggtggt catcatgcgc cagctttcat ccccgatatg
     5821 caccaccggg taaagttcac gggagacttt atctgacagc agacgtgcac tggccagggg
     5881 gatcaccatc cgtcgcccgg gcgtgtcaat aatatcactc tgtacatcca caaacagacg
     5941 ataacggctc tctcttttat aggtgtaaac cttaaactgc atttcaccag cccctgttct
     6001 cgtcagcaaa agagccgttc atttcaataa accgggcgac ctcagccatc ccttcctgat
     6061 tttccgcttt ccagcgttcg gcacgcagac gacgggcttc attctgcatg gttgtgctta
     6121 ccagaccgga gatattgaca tcatatatgc cttgagcaac tgatagctgt cgctgtcaac
     6181 tgtcactgta atacgctgct tcatagcata cctctttttg acatacttcg ggtatacata
     6241 tcagtatata ttcttatacc gcaaaaatca gcgcgcaaat acgcatactg ttatctggct
     6301 tttagtaagc cggatccacg cgtttacgcc ccgccctgcc actcatcgca gtactgttgt
     6361 aattcattaa gcattctgcc gacatggaag ccatcacaaa cggcatgatg aacctgaatc
     6421 gccagcggca tcagcacctt gtcgccttgc gtataatatt tgcccatggt gaaaacgggg
     6481 gcgaagaagt tgtccatatt ggccacgttt aaatcaaaac tggtgaaact cacccaggga
     6541 ttggctgaga cgaaaaacat attctcaata aaccctttag ggaaataggc caggttttca
     6601 ccgtaacacg ccacatcttg cgaatatatg tgtagaaact gccggaaatc gtcgtggtat
     6661 tcactccaga gcgatgaaaa cgtttcagtt tgctcatgga aaacggtgta acaagggtga
     6721 acactatccc atatcaccag ctcaccgtct ttcattgcca tacggaattc cggatgagca
     6781 ttcatcaggc gggcaagaat gtgaataaag gccggataaa acttgtgctt atttttcttt
     6841 acggtcttta aaaaggccgt aatatccagc tgaacggtct ggttata
//
