LOCUS       sequence-267862-        5133 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses HIV Tat for efficient sgRNA packaging. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5133)
  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 5133)
  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: pcDNA1-Tat
FEATURES             Location/Qualifiers
     source          1..5133
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        380..583
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     534..554
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     628..647
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        628..646
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(1096..1114)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1097..1114)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     intron          1198..1263
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             1393..1413
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    1685..1819
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1722..1741)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1776..1795
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      2665..2800
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3295..3314
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     rep_origin      complement(3427..3885)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3564..3585)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3779..3798
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(4009..4027)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4095..4199
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4200..4268,4269..5060)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4418..4437)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     enhancer        join(5133,1..379)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 acattgatta ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc
       61 atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa
      121 cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac
      181 tttccattga cgtcaatggg tggactattt acggtaaact gcccacttgg cagtacatca
      241 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg
      301 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt
      361 agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg
      421 gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg
      481 gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat
      541 gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctctctggc taactagaga
      601 acccactgct tactggctta tcgaaattaa tacgactcac tatagggaga cccaagcttg
      661 tcgacatagc agaataggcg ttactcaaca gaggagagca agaaatggag ccagtagatc
      721 ctagactaga gccctggaag catccaggaa gtcagcctaa aactgcttgt accacttgct
      781 attgtaaaaa gtgttgcttt cattgccaag tttgtttcac aacaaaagcc ttaggcatct
      841 cctatggcag gaagaagcgg agacagcgac gaagacctcc tcaaggcagt cagactcatc
      901 aagtttctct atcaaagcag taagtagtac atgtaatgca acctatacaa atagcaatag
      961 cagcattagt agtagcaata ataatagcaa tagttgtgtg gtccggatcc actagtaacg
     1021 gccgccagtg tgctggaatt ctgcagatat ccatcacact ggcggccgct cgagcatgca
     1081 tctagagggc cctattctat agtgtcacct aaatgctaga ggatctttgt gaaggaacct
     1141 tacttctgtg gtgtgacata attggacaaa ctacctacag agatttaaag ctctaaggta
     1201 aatataaaat ttttaagtgt ataatgtgtt aaactactga ttctaattgt ttgtgtattt
     1261 tagattccaa cctatggaac tgatgaatgg gagcagtggt ggaatgcctt taatgaggaa
     1321 aacctgtttt gctcagaaga aatgccatct agtgatgatg aggctactgc tgactctcaa
     1381 cattctactc ctccaaaaaa gaagagaaag gtagaagacc ccaaggactt tccttcagaa
     1441 ttgctaagtt ttttgagtca tgctgtgttt agtaatagaa ctcttgcttg ctttgctatt
     1501 tacaccacaa aggaaaaagc tgcactgcta tacaagaaaa ttatggaaaa atatttgatg
     1561 tatagtgcct tgactagaga tcataatcag ccataccaca tttgtagagg ttttacttgc
     1621 tttaaaaaac ctcccacacc tccccctgaa cctgaaacat aaaatgaatg caattgttgt
     1681 tgttaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     1741 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     1801 atcttatcat gtctggatca tcccgccatg gtatcaacgc catatttcta tttacagtag
     1861 ggacctcttc gttgtgtagg taccgctgta ttcctaggga aatagtagag gcaccttgaa
     1921 ctgtctgcat cagccatata gcccccgctg ttcgacttac aaacacaggc acagtactga
     1981 caaacccata cacctcctct gaaataccca tagttgctag ggctgtctcc gaactcatta
     2041 caccctccaa agtcagagct gtaatttcgc catcaagggc agcgagggct tctccagata
     2101 aaatagcttc tgccgagagt cccgtaaggg tagacacttc agctaatccc tcgatgaggt
     2161 ctactagaat agtcagtgcg gctcccattt tgaaaattca cttacttgat cagcttcaga
     2221 agatggcgga gggcctccaa cacagtaatt ttcctcccga ctcttaaaat agaaaatgtc
     2281 aagtcagtta agcaggaagt ggactaactg acgcagctgg ccgtgcgaca tcctctttta
     2341 attagttgct aggcaacgcc ctccagaggg cgtgtggttt tgcaagagga agcaaaagcc
     2401 tctccaccca ggcctagaat gtttccaccc aatcattact atgacaacag ctgttttttt
     2461 tagtattaag cagaggccgg ggacccctgg gcccgcttac tctggagaaa aagaagagag
     2521 gcattgtaga ggcttccaga ggcaacttgt caaaacagga ctgcttctat ttctgtcaca
     2581 ctgtctggcc ctgtcacaag gtccagcacc tccatacccc ctttaataag cagtttggga
     2641 acgggtgcgg gtcttactcc gcccatcccg cccctaactc cgcccagttc cgcccattct
     2701 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct
     2761 gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagcta
     2821 attcggcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     2881 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     2941 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     3001 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     3061 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     3121 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     3181 atacctacag cgtgagcatt gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     3241 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     3301 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     3361 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcaa gctagcttct
     3421 agctagaaat tgtaaacgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca
     3481 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaatagc
     3541 ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg
     3601 actccaacgt caaagggcga aaaaccgtct atcagggcga tggccgccca ctacgtgaac
     3661 catcacccaa atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta
     3721 aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag
     3781 ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg
     3841 taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtactat ggttgctttg
     3901 acgagcacgt ataacgtgct ttcctcgttg gaatcagagc gggagctaaa caggaggccg
     3961 attaaaggga ttttagacag gaacggtacg ccagctggac ccgaaagggc ctcgtgatac
     4021 gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt
     4081 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     4141 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     4201 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     4261 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     4321 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     4381 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     4441 gtgttgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     4501 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     4561 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     4621 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     4681 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     4741 ctgcagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     4801 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     4861 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     4921 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     4981 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     5041 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     5101 taaaacttca tttttaattt ctctagcgcg ttg
//
