LOCUS       sequence-319370-        7169 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  human CD63-TEV cleavage site-tTA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7169)
  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 7169)
  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: pcDNA3.1-CD63-(TEV)-tTA
FEATURES             Location/Qualifiers
     source          1..7169
                     /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"
     regulatory      752..761
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             758..1471
                     /codon_start=1
                     /product="human CD63 tetraspanin"
                     /label=CD63
                     /note="enriched in intraluminal vesicles of late endosomes 
                     (multivesicular bodies) and in exosomes"
                     /translation="MAVEGGMKCVKFLLYVLLLAFCACAVGLIAVGVGAQLVLSQTIIQ
                     GATPGSLLPVVIIAVGVFLFLVAFVGCCGACKENYCLMITFAIFLSLIMLVEVAAAIAG
                     YVFRDKVMSEFNNNFRQQMENYPKNNHTASILDRMQADFKCCGAANYTDWEKIPSMSKN
                     RVPDSCCINVTVGCGINFNEKAIHKEGCVEKIGGWLRKNVLVVAAAALGIAFVEVLGIV
                     FACCLVKSIRSGYEVM"
     CDS             1472..1492
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             join(1490..2107,2108..2494)
                     /codon_start=1
                     /product="tetracycline-controlled transactivator, 
                     comprising a fusion of the tetracycline repressor TetR with
                     the C-terminal activation domain of herpes simplex virus 
                     VP16"
                     /label=tTA
                     /note="In the Tet-Off(R) system, tTA binds to the 
                     Tet-responsive element and stimulates transcription only in
                     the absence of tetracycline or doxycycline."
                     /translation="SRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHVK
                     NKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTRP
                     TEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTTD
                     SMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGSAYSRARTKNNYGSTIE
                     GLLDLPDDDAPEEAGLAAPRLSFLPAGHTRRLSTAPPTDVSLGDELHLDGEDVAMAHAD
                     ALDDFDLDMLGDGDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALGIDEYGG"
     primer_bind     complement(1560..1579)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     complement(2526..2543)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2532..2756
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2802..3230
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2889..2908)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3099..3120
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3239..3259)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3244..3573
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3424..3559
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3486..3505
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3640..4434
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3694..3713)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4304..4323
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4608..4729
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4645..4664)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4699..4718
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4778..4794)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4778..4794)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4791..4813)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4802..4818
                     /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(4826..4832,4833..4850,4851..4856))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4871..4892
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5009..5026)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5180..5768)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5260..5279)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5939..6730,6731..6799))
                     /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     6562..6581
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6800..6904)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6979..6998)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
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 acccaagctg
      661 gctagcgttt aaacttaagc ttggtaccga gctcggatcc actagtccag tgtggtggaa
      721 ttctgcagat atccagcaca gtggcggccg cgccaccatg gcggtggaag gaggaatgaa
      781 atgtgtgaag ttcttgctct acgtcctcct gctggccttt tgcgcctgtg cagtgggact
      841 gattgccgtg ggtgtcgggg cacagcttgt cctgagtcag accataatcc agggggctac
      901 ccctggctct ctgttgccag tggtcatcat cgcagtgggt gtcttcctct tcctggtggc
      961 ttttgtgggc tgctgcgggg cctgcaagga gaactattgt cttatgatca cgtttgccat
     1021 ctttctgtct cttatcatgt tggtggaggt ggccgcagcc attgctggct atgtgtttag
     1081 agataaggtg atgtcagagt ttaataacaa cttccggcag cagatggaga attacccgaa
     1141 aaataaccac actgcttcga tcctggacag gatgcaggca gattttaagt gctgtggggc
     1201 tgctaactac acagattggg agaaaatccc ttccatgtcg aagaaccgag tccccgactc
     1261 ctgctgcatt aatgttactg tgggctgtgg gattaatttc aacgagaagg cgatccataa
     1321 ggagggctgt gtggagaaga ttgggggctg gctgaggaaa aatgtgctgg tggtagctgc
     1381 agcagccctt ggaattgctt ttgtcgaggt tttgggaatt gtctttgcct gctgcctcgt
     1441 gaagagtatc agaagtggct acgaggtgat ggagaacctg tacttccagt ctagattaga
     1501 taaaagtaaa gtgattaaca gcgcattaga gctgcttaat gaggtcggaa tcgaaggttt
     1561 aacaacccgt aaactcgccc agaagctagg tgtagagcag cctacattgt attggcatgt
     1621 aaaaaataag cgggctttgc tcgacgcctt agccattgag atgttagata ggcaccatac
     1681 tcacttttgc cctttagaag gggaaagctg gcaagatttt ttacgtaata acgctaaaag
     1741 ttttagatgt gctttactaa gtcatcgcga tggagcaaaa gtacatttag gtacacggcc
     1801 tacagaaaaa cagtatgaaa ctctcgaaaa tcaattagcc tttttatgcc aacaaggttt
     1861 ttcactagag aatgcattat atgcactcag cgctgtgggg cattttactt taggttgcgt
     1921 attggaagat caagagcatc aagtcgctaa agaagaaagg gaaacaccta ctactgatag
     1981 tatgccgcca ttattacgac aagctatcga attatttgat caccaaggtg cagagccagc
     2041 cttcttattc ggccttgaat tgatcatatg cggattagaa aaacaactta aatgtgaaag
     2101 tgggtccgcg tacagccgcg cgcgtacgaa aaacaattac gggtctacca tcgagggcct
     2161 gctcgatctc ccggacgacg acgcccccga agaggcgggg ctggcggctc cgcgcctgtc
     2221 ctttctcccc gcgggacaca cgcgcagact gtcgacggcc cccccgaccg atgtcagcct
     2281 gggggacgag ctccacttag acggcgagga cgtggcgatg gcgcatgccg acgcgctaga
     2341 cgatttcgat ctggacatgt tgggggacgg ggattccccg ggtccgggat ttacccccca
     2401 cgactccgcc ccctacggcg ctctggatat ggccgacttc gagtttgagc agatgtttac
     2461 cgatgccctt ggaattgacg agtacggtgg gtagtctaga gggcccgttt aaacccgctg
     2521 atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc
     2581 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc
     2641 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa
     2701 gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc
     2761 tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc
     2821 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     2881 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     2941 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     3001 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     3061 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     3121 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
     3181 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt aattctgtgg
     3241 aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     3301 agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc
     3361 agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg
     3421 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt
     3481 ttttttattt atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga
     3541 ggaggctttt ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt
     3601 ttcggatctg atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg
     3661 cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag
     3721 acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt
     3781 tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta
     3841 tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg
     3901 ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt
     3961 gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat
     4021 ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg
     4081 atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca
     4141 gccgaactgt tcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc
     4201 catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc
     4261 gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat
     4321 attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc
     4381 gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga
     4441 ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt
     4501 ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga
     4561 tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg
     4621 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     4681 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta
     4741 taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     4801 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     4861 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     4921 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     4981 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     5041 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     5101 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     5161 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     5221 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     5281 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     5341 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     5401 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     5461 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     5521 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     5581 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     5641 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     5701 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     5761 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     5821 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     5881 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     5941 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     6001 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     6061 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     6121 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     6181 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     6241 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     6301 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     6361 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     6421 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     6481 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     6541 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     6601 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     6661 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     6721 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     6781 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     6841 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     6901 cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcgggagat ctcccgatcc
     6961 cctatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatctgct
     7021 ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag
     7081 gcaaggcttg accgacaatt gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct
     7141 tcgcgatgta cgggccagat atacgcgtt
//
