LOCUS       sequence-275568-        5138 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 5138)
  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 5138)
  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..5138
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(1097..1115)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1098..1115)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     intron          1199..1264
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             1394..1414
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    1686..1820
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1723..1742)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1777..1796
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      2671..2806
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3301..3320
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     rep_origin      complement(3433..3891)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3570..3591)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3785..3804
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(4015..4033)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4101..4205
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4206..4274,4275..5066)
                     /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(4424..4443)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctt
      661 gtcgacatag cagaataggc gttactcaac agaggagagc aagaaatgga gccagtagat
      721 cctagactag agccctggaa gcatccagga agtcagccta aaactgcttg taccacttgc
      781 tattgtaaaa agtgttgctt tcattgccaa gtttgtttca caacaaaagc cttaggcatc
      841 tcctatggca ggaagaagcg gagacagcga cgaagacctc ctcaaggcag tcagactcat
      901 caagtttctc tatcaaagca gtaagtagta catgtaatgc aacctataca aatagcaata
      961 gcagcattag tagtagcaat aataatagca atagttgtgt ggtccggatc cactagtaac
     1021 ggccgccagt gtgctggaat tctgcagata tccatcacac tggcggccgc tcgagcatgc
     1081 atctagaggg ccctattcta tagtgtcacc taaatgctag aggatctttg tgaaggaacc
     1141 ttacttctgt ggtgtgacat aattggacaa actacctaca gagatttaaa gctctaaggt
     1201 aaatataaaa tttttaagtg tataatgtgt taaactactg attctaattg tttgtgtatt
     1261 ttagattcca acctatggaa ctgatgaatg ggagcagtgg tggaatgcct ttaatgagga
     1321 aaacctgttt tgctcagaag aaatgccatc tagtgatgat gaggctactg ctgactctca
     1381 acattctact cctccaaaaa agaagagaaa ggtagaagac cccaaggact ttccttcaga
     1441 attgctaagt tttttgagtc atgctgtgtt tagtaataga actcttgctt gctttgctat
     1501 ttacaccaca aaggaaaaag ctgcactgct atacaagaaa attatggaaa aatatttgat
     1561 gtatagtgcc ttgactagag atcataatca gccataccac atttgtagag gttttacttg
     1621 ctttaaaaaa cctcccacac ctccccctga acctgaaaca taaaatgaat gcaattgttg
     1681 ttgttaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     1741 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     1801 tatcttatca tgtctggatc gatcccgcca tggtatcaac gccatatttc tatttacagt
     1861 agggacctct tcgttgtgta ggtaccgctg tattcctagg gaaatagtag aggcaccttg
     1921 aactgtctgc atcagccata tagcccccgc tgttcgattt acaaacacag gcacagtact
     1981 gacaaaccca tacacctcct ctgaaatacc catagttgct agggctgtct ccgaactcat
     2041 tacaccctcc aaagtcagag ctgtaatttc gccatcaagg gcagcgaggg cttctccaga
     2101 taaaatagct tctgccgaga gtcccgtaag ggtagacact tcagctaatc cctcgatgag
     2161 gtctactaga atagtcagtg cggctcccat tttgaaaatt cacttacttg atcagcttca
     2221 gaagatggcg gagggcctcc aacacagtaa ttttcctccc gactcttaaa atagaaaatg
     2281 tcaagtcagt taagcaggaa gtgactaact gaccgcagct ggccgtgcga catcctcttt
     2341 taattagttg ctaggcaact gccctccaga gggcagtgtg gttttgcaag aggaagcaaa
     2401 aagcctctcc acccaggcct agaatgtttc cacccaatca ttactatgac aacagctgtt
     2461 ttttttagta ttaagcagag gccgggggcc cctggcctcc gcttactctg gagaaaaaga
     2521 agagaggcat tgtagaggct tccagaggca acttgtcaaa acaggactgc ttctatttct
     2581 gtcacactgt ctggccctgt cacaaggtcc agcacctcca tacccccttt aataagcagt
     2641 ttgggaacgg gtgcgggtct tactccgccc atcccgcccc taactccgcc cagttccgcc
     2701 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg
     2761 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa
     2821 aagctaattc ggcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     2881 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     2941 gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     3001 tgtagcaccg cctacatacc tcgctctgct gaagccagtt accagtggct gctgccagtg
     3061 gcgataagtc gtgtcttacc gggttggact caagagatag ttaccggata aggcgcagcg
     3121 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     3181 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     3241 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     3301 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     3361 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcaagcta
     3421 gcttctagct agaaattgta aacgttaata ttttgttaaa attcgcgtta aatttttgtt
     3481 aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag
     3541 aatagcccga gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga
     3601 acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc cgcccactac
     3661 gtgaaccatc acccaaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga
     3721 accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa
     3781 aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc
     3841 tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg tactatggtt
     3901 gctttgacga gcacgtataa cgtgctttcc tcgttagaat cagagcggga gctaaacagg
     3961 aggccgatta aagggatttt agacaggaac ggtacgccag ctggacccga aagggcctcg
     4021 tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg
     4081 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     4141 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     4201 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     4261 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     4321 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     4381 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     4441 tatcccgtgt tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     4501 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     4561 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     4621 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     4681 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     4741 cgatgcctgc agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     4801 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     4861 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     4921 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     4981 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     5041 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     5101 ttgatttaaa acttcatttt taatttctct agcgcgtt
//
