LOCUS       Exported                4668 bp ds-DNA     circular SYN 07-SEP-2021
DEFINITION  Expression of GFP-tagged, dominant negative VPS4 mutant.
ACCESSION   .
VERSION     .
KEYWORDS    pEGFP-VSP4-E228Q
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4668)
  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 4668)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Wednesday, Dec 11, 2024 from SnapGene 5.0.8
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..4668
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     545..566
                     /label=CS174
     primer_bind     581..633
                     /label=CS490_AgeImTagBFP2_F60.6
     regulatory      607..616
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             613..1323
                     /codon_start=1
                     /product="enhanced monomeric blue fluorescent protein 
                     (Subach et al., 2011)"
                     /label=mTagBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRND
                     MALKLVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAV
                     ARYCDLPSKLGHKLN"
     primer_bind     complement(1305..1351)
                     /label=CS605_endBFPbam_R61
     CDS             1324..1326
                     /codon_start=1
                     /label=VPS4
                     /translation=""
     primer_bind     complement(1330..1350)
                     /label=CS391
     polyA_signal    1456..1577
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1493..1512)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1547..1566
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1584..2039)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1721..1742)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1933..1952
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2066..2170
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2172..2529
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     primer_bind     complement(2195..2215)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2380..2515
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2442..2461
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     complement(2465..2476)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2564..3358
                     /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(2618..2637)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3228..3247
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3546..3565)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3590..3637
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      3966..4554
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4455..4474
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      601 ccggtcgcca ccatggtgtc taagggcgaa gagctgatta aggagaacat gcacatgaag
      661 ctgtacatgg agggcaccgt ggacaaccat cacttcaagt gcacatccga gggcgaaggc
      721 aagccctacg agggcaccca gaccatgaga atcaaggtgg tcgagggcgg ccctctcccc
      781 ttcgccttcg acatcctggc tactagcttc ctctacggca gcaagacctt catcaaccac
      841 acccagggca tccccgactt cttcaagcag tccttccctg agggcttcac atgggagaga
      901 gtcaccacat acgaagacgg gggcgtgctg accgctaccc aggacaccag cctccaggac
      961 ggctgcctca tctacaacgt caagatcaga ggggtgaact tcacatccaa cggccctgtg
     1021 atgcagaaga aaacactcgg ctgggaggcc ttcaccgaga cgctgtaccc cgctgacggc
     1081 ggcctggaag gcagaaacga catggccctg aagctcgtgg gcgggagcca tctgatcgca
     1141 aacgccaaga ccacatatag atccaagaaa cccgctaaga acctcaagat gcctggcgtc
     1201 tactatgtgg actacagact ggaaagaatc aaggaggcca acaacgagac ctacgtcgag
     1261 cagcacgagg tggcagtggc cagatactgc gacctcccta gcaaactggg gcacaagctt
     1321 aattaaggat ccaccggatc tagataactg atcataatca gccataccac atttgtagag
     1381 gttttacttg ctttaaaaaa cctcccacac ctccccctga acctgaaaca taaaatgaat
     1441 gcaattgttg ttgttaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     1501 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     1561 ctcatcaatg tatcttaacg cgtaaattgt aagcgttaat attttgttaa aattcgcgtt
     1621 aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta
     1681 taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc
     1741 actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg
     1801 cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact
     1861 aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt
     1921 ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc
     1981 ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc
     2041 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     2101 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     2161 aaggaagagt cctgaggcgg aaagaaccag ctgtggaatg tgtgtcagtt agggtgtgga
     2221 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     2281 accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     2341 aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc
     2401 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
     2461 gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc
     2521 ttttgcaaag atcgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg
     2581 attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca
     2641 acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt
     2701 tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaagacg aggcagcgcg
     2761 gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga
     2821 agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca
     2881 ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct
     2941 tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac
     3001 tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc
     3061 gccagccgaa ctgttcgcca ggctcaaggc gagcatgccc gacggcgagg atctcgtcgt
     3121 gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt
     3181 catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg
     3241 tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat
     3301 cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc
     3361 gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc
     3421 gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc
     3481 tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc tagggggagg
     3541 ctaactgaaa cacggaagga gacaataccg gaaggaaccc gcgctatgac ggcaataaaa
     3601 agacagaata aaacgcacgg tgttgggtcg tttgttcata aacgcggggt tcggtcccag
     3661 ggctggcact ctgtcgatac cccaccgaga ccccattggg gccaatacgc ccgcgtttct
     3721 tccttttccc caccccaccc cccaagttcg ggtgaaggcc cagggctcgc agccaacgtc
     3781 ggggcggcag gccctgccat agcctcaggt tactcatata tactttagat tgatttaaaa
     3841 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     3901 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     3961 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     4021 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     4081 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
     4141 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     4201 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     4261 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     4321 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     4381 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     4441 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     4501 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     4561 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     4621 cctgcgttat cccctgattc tgtggataac cgtattaccg ccatgcat
//
