LOCUS       sequence-319368-        4019 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  C-terminally PEST-tagged NanoLuc under TRE3G promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4019)
  AUTHORS   Somiya M, Kuroda S
  TITLE     Reporter gene assay for membrane fusion of extracellular vesicles.
  JOURNAL   J Extracell Vesicles. 2021 Nov;10(13):e12171. doi: 
            10.1002/jev2.12171.
  PUBMED    34807503
REFERENCE   2  (bases 1 to 4019)
  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: "J Extracell
            Vesicles."; date: "2021-11"; volume: "10(13):e12171. doi"; pages: " 
            10.1002/jev2.12171"
COMMENT     Sequence Label: pTRE3G-NlucP
FEATURES             Location/Qualifiers
     source          1..4019
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..379
                     /label=TRE3G promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G"
     protein_bind    9..27
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    45..63
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    81..99
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    117..135
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    153..171
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    189..207
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    225..243
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     288..312
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      385..394
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             391..903
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     CDS             907..1026
                     /codon_start=1
                     /product="PEST degradation sequence from mouse ornithine 
                     decarboxylase"
                     /label=hPEST
                     /note="human codon-optimized"
                     /translation="SHGFPPEVEEQAAGTLPMSCAQESGMDRHPAACASARINV"
     intron          1168..1233
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             1363..1383
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    1808..1929
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1845..1864)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1899..1918
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2015..2032)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2186..2774)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2266..2285)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2945..3736,3737..3805))
                     /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     3568..3587
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3806..3910)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3978..3996
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 gagtttactc cctatcagtg atagagaacg tatgaagagt ttactcccta tcagtgatag
       61 agaacgtatg cagactttac tccctatcag tgatagagaa cgtataagga gtttactccc
      121 tatcagtgat agagaacgta tgaccagttt actccctatc agtgatagag aacgtatcta
      181 cagtttactc cctatcagtg atagagaacg tatatccagt ttactcccta tcagtgatag
      241 agaacgtata agctttaggc gtgtacggtg ggcgcctata aaagcagagc tcgtttagtg
      301 aaccgtcaga tcgcctggag caattccaca acacttttgt cttataccaa ctttccgtac
      361 cacttcctac cctcgtaaag tcgagccacc atggtcttca cactcgaaga tttcgttggg
      421 gactggcgac agacagccgg ctacaacctg gaccaagtcc ttgaacaggg aggtgtgtcc
      481 agtttgtttc agaatctcgg ggtgtccgta actccgatcc aaaggattgt cctgagcggt
      541 gaaaatgggc tgaagatcga catccatgtc atcatcccgt atgaaggtct gagcggcgac
      601 caaatgggcc agatcgaaaa aatttttaag gtggtgtacc ctgtggatga tcatcacttt
      661 aaggtgatcc tgcactatgg cacactggta atcgacgggg ttacgccgaa catgatcgac
      721 tatttcggac ggccgtatga aggcatcgcc gtgttcgacg gcaaaaagat cactgtaaca
      781 gggaccctgt ggaacggcaa caaaattatc gacgagcgcc tgatcaaccc cgacggctcc
      841 ctgctgttcc gagtaaccat caacggagtg accggctggc ggctgtgcga acgcattctg
      901 gcgaattctc acggctttcc gcctgaggtt gaagagcaag ccgccggtac attgcctatg
      961 tcctgcgcac aagaaagcgg tatggaccgg cacccagccg cttgtgcttc agctcgcatc
     1021 aacgtctaat aagatccaat gtaactgtat tcagcgatga cgaaattctt agctattgta
     1081 atactctaga ggatctttgt gaaggaacct tacttctgtg gtgtgacata attggacaaa
     1141 ctacctacag agatttaaag ctctaaggta aatataaaat ttttaagtgt ataatgtgtt
     1201 aaactactga ttctaattgt ttgtgtattt tagattccaa cctatggaac tgatgaatgg
     1261 gagcagtggt ggaatgcctt taatgaggaa aacctgtttt gctcagaaga aatgccatct
     1321 agtgatgatg aggctactgc tgactctcaa cattctactc ctccaaaaaa gaagagaaag
     1381 gtagaagacc ccaaggactt tccttcagaa ttgctaagtt ttttgagtca tgctgtgttt
     1441 agtaatagaa ctcttgcttg ctttgctatt tacaccacaa aggaaaaagc tgcactgcta
     1501 tacaagaaaa ttatggaaaa atattctgta acctttataa gtaggcataa cagttataat
     1561 cataacatac tgttttttct tactccacac aggcatagag tgtctgctat taataactat
     1621 gctcaaaaat tgtgtacctt tagcttttta atttgtaaag gggttaataa ggaatatttg
     1681 atgtatagtg ccttgactag agatcataat cagccatacc acatttgtag aggttttact
     1741 tgctttaaaa aacctcccac acctccccct gaacctgaaa cataaaatga atgcaattgt
     1801 tgttgttaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
     1861 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
     1921 tgtatcttat catgtctgcg gctctagagc tgcattaatg aatcggccaa cgcgcgggga
     1981 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     2041 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     2101 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     2161 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     2221 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     2281 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     2341 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     2401 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     2461 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     2521 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     2581 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     2641 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     2701 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     2761 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     2821 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     2881 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     2941 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     3001 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     3061 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     3121 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     3181 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     3241 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     3301 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     3361 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     3421 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     3481 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     3541 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     3601 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     3661 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     3721 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     3781 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     3841 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     3901 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca
     3961 tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtcttcaa gaattcctc
//
