LOCUS       sequence-454376-        6024 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of GFP-tagged, dominant negative VPS4 mutant.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6024)
  AUTHORS   Votteler J, Ogohara C, Yi S, Hsia Y, Nattermann U, Belnap DM, King 
            NP, Sundquist WI
  TITLE     Designed proteins induce the formation of nanocage-containing 
            extracellular vesicles.
  JOURNAL   Nature. 2016 Dec 8;540(7632):292-295. doi: 10.1038/nature20607. Epub
            2016 Nov 30.
  PUBMED    27919066
REFERENCE   2  (bases 1 to 6024)
  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"; doi: 
            "10.1038/nature20607"; journalName: "Nature"; date: "2016-12-8- 8"; 
            volume: "540"; issue: "7632"; pages: "292-295"
COMMENT     Sequence Label: pEGFP-VSP4-E228Q
FEATURES             Location/Qualifiers
     source          1..6024
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(825..1535,1536..1538,1539..1541))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(825..1535,1536..1538,1539..1541))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(867..888)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1216..1235
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1475..1496
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      1538..1547
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(1586..1789)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(1615..1635)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        1790..2093
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     rep_origin      complement(2268..2856)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2348..2367)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    3185..3232
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     3257..3276
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     CDS             complement(3464..4258)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             complement(3464..4258)
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3575..3594)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4185..4204
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(4293..4650)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4307..4442
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        4459..4650
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     4607..4627
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        complement(4652..4756)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4783..5238
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4870..4889)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5080..5101
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    5245..5366
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5256..5275)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5310..5329
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
ORIGIN
        1 ttgttattcc ccaccccctg catctggacc aagaactccg ttttgatcct ccgggcggcc
       61 tcactctcat tttcatttcg ggacccgcag agggaatcca cctggtcgat gaagatgatg
      121 gagggcttgt gctgcctggc cagctcaaac aggttcttga ccagcttttc actctccccc
      181 agccacttgg acatcagatc tgaggaggac acagagaaga aggtggagtt gttggcctct
      241 gttgccacgg ctttggccag gtaggatttc cctgtgccag ggggtccgaa cagcagaatc
      301 ccccgccagg gggtgcgctt gcctgtgaac aagtgtggga atttgattgg caaaatgaca
      361 gcttctttga gggcctcctt ggccccctcc agcccggcca cgtcgttcca ccgtatgttg
      421 ggcttctcca tcacgacggc acccatcagc tgttcttgca gtttcttttt ctccggatta
      481 tccccttcac tgtcactgtc actgcccttg ccctcactct ggttctcttt gactggcttc
      541 ttgccgtgtt tctctttgct tcgtaaataa tccttcagct tctcggcccg gtctaggtac
      601 tgcacgcact tggctcgaat gctctccttg gccttgtcgc tgtgggcctc atacttgata
      661 gcgtggagga agtactccac cgcatgctgg tacagccgca gcgcctcctc gtagttcttg
      721 gctttgtcct cctctgtggc tttcgtcacc agatcaatgg ctttctggag ggttgacgtt
      781 gtcatcgcag aattcgaagc ttgagctcga gatctgagtc cggacttgta cagctcgtcc
      841 atgccgagag tgatcccggc ggcggtcacg aactccagca ggaccatgtg atcgcgcttc
      901 tcgttggggt ctttgctcag ggcggactgg gtgctcaggt agtggttgtc gggcagcagc
      961 acggggccgt cgccgatggg ggtgttctgc tggtagtggt cggcgagctg cacgctgccg
     1021 tcctcgatgt tgtggcggat cttgaagttc accttgatgc cgttcttctg cttgtcggcc
     1081 atgatataga cgttgtggct gttgtagttg tactccagct tgtgccccag gatgttgccg
     1141 tcctccttga agtcgatgcc cttcagctcg atgcggttca ccagggtgtc gccctcgaac
     1201 ttcacctcgg cgcgggtctt gtagttgccg tcgtccttga agaagatggt gcgctcctgg
     1261 acgtagcctt cgggcatggc ggacttgaag aagtcgtgct gcttcatgtg gtcggggtag
     1321 cggctgaagc actgcacgcc gtaggtcagg gtggtcacga gggtgggcca gggcacgggc
     1381 agcttgccgg tggtgcagat gaacttcagg gtcagcttgc cgtaggtggc atcgccctcg
     1441 ccctcgccgg acacgctgaa cttgtggccg tttacgtcgc cgtccagctc gaccaggatg
     1501 ggcaccaccc cggtgaacag ctcctcgccc ttgctcacca tggtggcgac cggtagcgct
     1561 agcggatctg acggttcact aaaccagctc tgcttatata gacctcccac cgtacacgcc
     1621 taccgcccat ttgcgtcaat ggggcggagt tgttacgaca ttttggaaag tcccgttgat
     1681 tttggtgcca aaacaaactc ccattgacgt caatggggtg gagacttgga aatccccgtg
     1741 agtcaaaccg ctatccacgc ccattgatgt actgccaaaa ccgcatcacc atggtaatag
     1801 cgatgactaa tacgtagatg tactgccaag taggaaagtc ccataaggtc atgtactggg
     1861 cataatgcca ggcgggccat ttaccgtcat tgacgtcaat agggggcgta cttggcatat
     1921 gatacacttg atgtactgcc aagtgggcag tttaccgtaa atactccacc cattgacgtc
     1981 aatggaaagt ccctattggc gttactatgg gaacatacgt cattattgac gtcaatgggc
     2041 gggggtcgtt gggcggtcag ccaggcgggc catttaccgt aagttatgta acgcggaact
     2101 ccatatatgg gctatgaact aatgaccccg taattgatta ctattaataa ctaatgcatg
     2161 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     2221 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     2281 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     2341 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     2401 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     2461 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     2521 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     2581 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     2641 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     2701 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     2761 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     2821 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     2881 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     2941 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     3001 atatatgagt aacctgaggc tatggcaggg cctgccgccc cgacgttggc tgcgagccct
     3061 gggccttcac ccgaacttgg ggggtggggt ggggaaaagg aagaaacgcg ggcgtattgg
     3121 ccccaatggg gtctcggtgg ggtatcgaca gagtgccagc cctgggaccg aaccccgcgt
     3181 ttatgaacaa acgacccaac accgtgcgtt ttattctgtc tttttattgc cgtcatagcg
     3241 cgggttcctt ccggtattgt ctccttccgt gtttcagtta gcctccccct agggtgggcg
     3301 aagaactcca gcatgagatc cccgcgctgg aggatcatcc agccggcgtc ccggaaaacg
     3361 attccgaagc ccaacctttc atagaaggcg gcggtggaat cgaaatctcg tgatggcagg
     3421 ttgggcgtcg cttggtcggt catttcgaac cccagagtcc cgctcagaag aactcgtcaa
     3481 gaaggcgata gaaggcgatg cgctgcgaat cgggagcggc gataccgtaa agcacgagga
     3541 agcggtcagc ccattcgccg ccaagctctt cagcaatatc acgggtagcc aacgctatgt
     3601 cctgatagcg gtccgccaca cccagccggc cacagtcgat gaatccagaa aagcggccat
     3661 tttccaccat gatattcggc aagcaggcat cgccatgggt cacgacgaga tcctcgccgt
     3721 cgggcatgct cgccttgagc ctggcgaaca gttcggctgg cgcgagcccc tgatgctctt
     3781 cgtccagatc atcctgatcg acaagaccgg cttccatccg agtacgtgct cgctcgatgc
     3841 gatgtttcgc ttggtggtcg aatgggcagg tagccggatc aagcgtatgc agccgccgca
     3901 ttgcatcagc catgatggat actttctcgg caggagcaag gtgagatgac aggagatcct
     3961 gccccggcac ttcgcccaat agcagccagt cccttcccgc ttcagtgaca acgtcgagca
     4021 cagctgcgca aggaacgccc gtcgtggcca gccacgatag ccgcgctgcc tcgtcttgca
     4081 gttcattcag ggcaccggac aggtcggtct tgacaaaaag aaccgggcgc ccctgcgctg
     4141 acagccggaa cacggcggca tcagagcagc cgattgtctg ttgtgcccag tcatagccga
     4201 atagcctctc cacccaagcg gccggagaac ctgcgtgcaa tccatcttgt tcaatcatgc
     4261 gaaacgatcc tcatcctgtc tcttgatcga tctttgcaaa agcctaggcc tccaaaaaag
     4321 cctcctcact acttctggaa tagctcagag gccgaggcgg cctcggcctc tgcataaata
     4381 aaaaaaatta gtcagccatg gggcggagaa tgggcggaac tgggcggagt taggggcggg
     4441 atgggcggag ttaggggcgg gactatggtt gctgactaat tgagatgcat gctttgcata
     4501 cttctgcctg ctggggagcc tggggacttt ccacacctgg ttgctgacta attgagatgc
     4561 atgctttgca tacttctgcc tgctggggag cctggggact ttccacaccc taactgacac
     4621 acattccaca gctggttctt tccgcctcag gactcttcct ttttcaatat tattgaagca
     4681 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     4741 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgcgccc tgtagcggcg
     4801 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     4861 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     4921 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     4981 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     5041 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     5101 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
     5161 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
     5221 tattaacgct tacaatttac gcgttaagat acattgatga gtttggacaa accacaacta
     5281 gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa
     5341 ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg
     5401 ttcaggggga ggtgtgggag gttttttaaa gcaagtaaaa cctctacaaa tgtggtatgg
     5461 ctgattatga tcagttatct agatccggtg gatccttaac tctcttgccc aaagtcctct
     5521 gagaatttct tcactttcag gaggtcgtct gcattcaccg tgggccgggt ggtggccaga
     5581 gaccgcagca tgtccgacat gcaaaccaca ggctctaaga gtttgtcccc agggacatcc
     5641 atccaagtca tctccatggc tcctgggtcc cctggtgagc atggagtcag gaggtcatca
     5701 atcatcatgc tggggttggt gcgagagggg ccacagacct ttttgaagtg tgtggccgac
     5761 tgcaccttcc tcacgggctg catgagagag tcccgcacga tgatgctgat gtccgcgccc
     5821 gagtagcctt ccgtcttccg ggccagctcg tggatgtttg catccgtgag gttgtgggga
     5881 gtgctcccga gatgcaaccg gaacatctgg gcgcgggcag cttcctccgg caaggggata
     5941 taaattcgct tttcaaacct cctcctgatg gccgaatcca acacccatgg gatgtttgtg
     6001 gctccaagaa ccagagtccc atca
//
