LOCUS       Exported                6672 bp ds-DNA     circular SYN 19-JAN-2024
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 6672)
  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 6672)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Friday, Jan 19, 2024 from SnapGene 5.0.8
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6672
                     /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
     misc_feature    600..605
                     /label=AgeI
     regulatory      607..616
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     primer_bind     610..629
                     /label=CS458
     CDS             613..1329
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(658..679)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(919..938)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1203..1220
                     /label=CS20_eGFP_check_F58.7
     primer_bind     1266..1287
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1301..1317
                     /label=CS21_endGFP_F59.8
     primer_bind     complement(1310..1329)
                     /label=CS22
     CDS             1348..1956
                     /codon_start=1
                     /label=RNF152
                     /translation="METLSQDSLLECQICFNYYSPRRRPKLLDCKHTCCSVCLQQMRTS
                     QKDVRCPWCRGVTKLPPGFSVSQLPDDPEVLAVIAIPHTSEHTPVFIKLPSNGCYMLPL
                     PISKERALLPGDMGCRLLPGSQQKSVTVVTIPAEQQPLQGGAPQEAVEEEQDRRGVVKS
                     STWSGVCTVILVACVLVFLLGIVLHNMSCISKRFTVISCG"
     primer_bind     1893..1911
                     /label=CS642_RNF152chk_F60
     CDS             1957..1959
                     /codon_start=1
                     /label=VPS4
                     /translation=""
     primer_bind     1959..1977
                     /label=CS393_MCS_F62.3
     misc_feature    1960..2012
                     /label=MCS
                     /note="multiple cloning site of fluorescent protein 
                     plasmids"
     primer_bind     complement(1975..1992)
                     /label=CS455_IRESmcs_R59.5
     misc_feature    2014..2600
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     2109..2128
                     /label=CS468_IRESchk_F61.6
     primer_bind     2580..2617
                     /label=CS458
     primer_bind     complement(2580..2603)
                     /label=CS450_ires_R61.2
     CDS             2589..2591
                     /codon_start=1
                     /product="start codon for translation from IRES2"
                     /label=ATG
                     /translation="M"
     CDS             2601..3296
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet-I
                     /note="matures faster than mScarlet"
                     /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFSWDILSPQFMYGSRAFIKHPADIPDYYKQSFPEGFKWERVMNFEDGG
                     AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK
                     MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH
                     STGGMDELYK"
     regulatory      2601..2604
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     primer_bind     complement(2954..2973)
                     /label=Unique_Scarlet
     primer_bind     3082..3100
                     /label=cs338_61.6
     primer_bind     3082..3099
                     /label=CS310
     primer_bind     3275..3284
                     /label=CS21_endGFP_F59.8
     primer_bind     complement(3277..3295)
                     /label=CS22
     misc_feature    3331..3336
                     /label=BamHI
     primer_bind     complement(3334..3354)
                     /label=CS391
     polyA_signal    3460..3581
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3497..3516)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3551..3570
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3588..4043)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3725..3746)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3937..3956
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4070..4174
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4176..4533
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     primer_bind     complement(4199..4219)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4384..4519
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4446..4465
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     complement(4469..4480)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4568..5362
                     /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(4622..4641)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5232..5251
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5550..5569)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5594..5641
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5970..6558
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6459..6478
                     /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 ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
      661 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
      721 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
      781 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc
      841 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
      901 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
      961 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     1021 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac
     1081 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc
     1141 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     1201 cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc
     1261 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
     1321 ctgtacaaag gaggttcagg aggatccatg gagacgctca gtcaggatag cttgttggaa
     1381 tgccaaatat gtttcaacta ctatagtccc agacgaagac ccaagttgct ggactgtaag
     1441 catacctgtt gtagcgtatg tctccaacaa atgcggacga gtcagaaaga cgttcgatgc
     1501 ccatggtgca ggggcgtcac taagctccct ccgggttttt ctgtaagcca actgccagac
     1561 gacccagagg tgctcgctgt gatcgcgata ccacatacat cagagcacac gccagtattt
     1621 atcaaacttc ctagtaacgg ttgctacatg ttgcccctgc cgataagcaa agaacgagca
     1681 ctcctccctg gggatatggg atgcagattg ctgcccggca gtcagcaaaa atcagtcaca
     1741 gttgttacca tacccgcaga gcaacaacct ctccagggtg gcgccccgca ggaggcagtc
     1801 gaagaagagc aggatcggcg aggcgtagtg aagagttcta catggagtgg cgtttgtacc
     1861 gtaatactgg tagcttgcgt cctggttttc ctgctcggca tagttcttca caatatgtct
     1921 tgtattagta aacgattcac tgtcatctcc tgtgggtaac tcgagctcaa gcttcgaatt
     1981 ctgcagtcga cggtaccgcg ggcccgggat ccgcccctct ccctcccccc cccctaacgt
     2041 tactggccga agccgcttgg aataaggccg gtgtgcgttt gtctatatgt tattttccac
     2101 catattgccg tcttttggca atgtgagggc ccggaaacct ggccctgtct tcttgacgag
     2161 cattcctagg ggtctttccc ctctcgccaa aggaatgcaa ggtctgttga atgtcgtgaa
     2221 ggaagcagtt cctctggaag cttcttgaag acaaacaacg tctgtagcga ccctttgcag
     2281 gcagcggaac cccccacctg gcgacaggtg cctctgcggc caaaagccac gtgtataaga
     2341 tacacctgca aaggcggcac aaccccagtg ccacgttgtg agttggatag ttgtggaaag
     2401 agtcaaatgg ctctcctcaa gcgtattcaa caaggggctg aaggatgccc agaaggtacc
     2461 ccattgtatg ggatctgatc tggggcctcg gtgcacatgc tttacatgtg tttagtcgag
     2521 gttaaaaaaa cgtctaggcc ccccgaacca cggggacgtg gttttccttt gaaaaacacg
     2581 atgataatat ggccacaacc atggtgagca agggcgaggc agtgatcaag gagttcatgc
     2641 ggttcaaggt gcacatggag ggctccatga acggccacga gttcgagatc gagggcgagg
     2701 gcgagggccg cccctacgag ggcacccaga ccgccaagct gaaggtgacc aagggtggcc
     2761 ccctgccctt ctcctgggac atcctgtccc ctcagttcat gtacggctcc agggccttca
     2821 tcaagcaccc cgccgacatc cccgactact ataagcagtc cttccccgag ggcttcaagt
     2881 gggagcgcgt gatgaacttc gaggacggcg gcgccgtgac cgtgacccag gacacctccc
     2941 tggaggacgg caccctgatc tacaaggtga agctccgcgg caccaacttc cctcctgacg
     3001 gccccgtaat gcagaagaag acaatgggct gggaagcgtc caccgagcgg ttgtaccccg
     3061 aggacggcgt gctgaagggc gacattaaga tggccctgcg cctgaaggac ggcggccgct
     3121 acctggcgga cttcaagacc acctacaagg ccaagaagcc cgtgcagatg cccggcgcct
     3181 acaacgtcga ccgcaagttg gacatcacct cccacaacga ggactacacc gtggtggaac
     3241 agtacgaacg ctccgagggc cgccactcca ccggcggcat ggacgagctg tacaagtaag
     3301 aattctgcag tcgacggtac cgcgggcccg ggatccaccg gatctagata actgatcata
     3361 atcagccata ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc
     3421 ctgaacctga aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat
     3481 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     3541 cattctagtt gtggtttgtc caaactcatc aatgtatctt aacgcgtaaa ttgtaagcgt
     3601 taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata
     3661 ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt
     3721 tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg
     3781 aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt
     3841 ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc
     3901 ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg
     3961 cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct
     4021 taatgcgccg ctacagggcg cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     4081 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     4141 ataaatgctt caataatatt gaaaaaggaa gagtcctgag gcggaaagaa ccagctgtgg
     4201 aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     4261 agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc
     4321 agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg
     4381 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt
     4441 ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga
     4501 ggaggctttt ttggaggcct aggcttttgc aaagatcgat caagagacag gatgaggatc
     4561 gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag
     4621 gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg
     4681 gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa
     4741 tgaactgcaa gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc
     4801 agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc
     4861 ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga
     4921 tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa
     4981 acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct
     5041 ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgagcat
     5101 gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt
     5161 ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta
     5221 tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga
     5281 ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg
     5341 ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg
     5401 cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc
     5461 ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag
     5521 ttcttcgccc accctagggg gaggctaact gaaacacgga aggagacaat accggaagga
     5581 acccgcgcta tgacggcaat aaaaagacag aataaaacgc acggtgttgg gtcgtttgtt
     5641 cataaacgcg gggttcggtc ccagggctgg cactctgtcg ataccccacc gagaccccat
     5701 tggggccaat acgcccgcgt ttcttccttt tccccacccc accccccaag ttcgggtgaa
     5761 ggcccagggc tcgcagccaa cgtcggggcg gcaggccctg ccatagcctc aggttactca
     5821 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     5881 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     5941 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     6001 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     6061 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt
     6121 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     6181 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     6241 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     6301 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     6361 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     6421 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     6481 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     6541 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     6601 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     6661 accgccatgc at
//
