LOCUS       sequence-175372-        7062 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of full-length S. cerevisiae Vps4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7062)
  AUTHORS   Monroe N, Han H, Shen PS, Sundquist WI, Hill CP
  TITLE     Structural Basis of Protein Translocation by the Vps4-Vta1 AAA 
            ATPase.
  JOURNAL   Elife. 2017 Apr 5;6. pii: e24487. doi: 10.7554/eLife.24487.
  PUBMED    28379137
REFERENCE   2  (bases 1 to 7062)
  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: "Elife."; 
            date: "2017-04-5"; volume: "6. pii: e24487. doi"; pages: " 
            10.7554/eLife.24487"
COMMENT     Sequence Label: pET151-Vps4
FEATURES             Location/Qualifiers
     source          1..7062
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     106..125
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     209..228
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        209..227
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    228..252
                     /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)."
     RBS             267..289
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             300..317
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             318..359
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             360..380
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(1763..1781)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1777..1824
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(2062..2080)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2148..2252
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2253..2321,2322..3113)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2471..2490)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3284..3872
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3773..3792
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4026..4043
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4058..4197
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4123..4142)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4283..4305
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4299..4490)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5802..6884)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     6846..6865
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6885..6962)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
       61 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
      121 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
      181 gaggatcgag atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg
      241 gataacaatt cccctctaga aataattttg tttaacttta agaaggagat atacatatgc
      301 atcatcacca tcaccatggt aagcctatcc ctaaccctct cctcggtctc gattctacgg
      361 aaaacctgta ttttcaggga atgagcacgg gagatttttt aactaaggga atagaactgg
      421 ttcaaaaggc cattgacctg gacacagcca cgcagtatga ggaggcgtat acagcatact
      481 ataacggatt agattatttg atgttggctt tgaaatacga gaaaaaccct aagtcgaagg
      541 atttgataag agcgaagttt acagagtatc taaaccgtgc agagcagttg aaaaagcact
      601 tggaaagtga agaggcaaac gcggcaaaga aatctcctag cgctggtagt ggttccaacg
      661 gtggtaataa gaaaatctct caggaagagg gtgaggataa tggtggagaa gacaacaaaa
      721 agctaagagg tgccttatcg agtgctattt tgtctgagaa gcccaatgtt aagtgggaag
      781 acgtggctgg actggaagga gccaaagagg cccttaaaga agccgttatt ttacctgtca
      841 agttcccaca tttgttcaaa ggtaaccgta agcccacttc gggaatctta ttgtacggac
      901 caccaggtac aggtaaatca tatttggcaa aagcagtggc caccgaggcc aattctacct
      961 ttttcagtgt tagttccagt gatttggttt ctaagtggat gggtgaatct gaaaaacttg
     1021 tcaaacagtt atttgcgatg gcaagagaaa ataaaccttc tattattttt atagatgaag
     1081 tggatgcgct aacaggtacc agaggagaag gagagagcga agcaagtaga agaattaaaa
     1141 cagaattatt ggtacaaatg aatggtgttg ggaatgactc ccaaggtgtt ttggtccttg
     1201 gcgcaacaaa tatcccatgg caactggaca gtgccatcag gagaagattt gaaagaagaa
     1261 tatatatacc attaccagat ttggcggcaa gaaccaccat gttcgagatt aacgtcggcg
     1321 acactccatg cgtattgacc aaggaagatt atagaacttt aggtgcaatg actgaaggtt
     1381 attccgggag tgatattgct gtagtggtga aagatgcgct aatgcaaccg ataagaaaaa
     1441 ttcaaagcgc gacacatttc aaagatgtct ctacggagga cgacgaaaca agaaaactaa
     1501 caccatgctc tccaggtgat gatggcgcca tcgagatgag ttggacggat attgaagctg
     1561 atgaattaaa agaaccagat ctcaccataa aggatttctt aaaggctatc aaatcgacga
     1621 gacctaccgt gaatgaggat gacttgctga agcaagaaca gttcactaga gattttggtc
     1681 aagaaggtaa ctagcatatg aagggcgagc tcagatccgg ctgctaacaa agcccgaaag
     1741 gaagctgagt tggctgctgc caccgctgag caataactag cataacccct tggggcctct
     1801 aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggata tcccgcaaga
     1861 ggcccggcag taccggcata accaagccta tgcctacagc atccagggtg acggtgccga
     1921 ggatgacgat gagcgcattg ttagatttca tacacggtgc ctgactgcgt tagcaattta
     1981 actgtgataa actaccgcat taaagctagc ttatcgatga taagctgtca aacatgagaa
     2041 ttaattcttg aagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga
     2101 taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     2161 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     2221 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     2281 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     2341 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     2401 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     2461 ttaaagttct gctatgtggc gcggtattat cccgtgttga cgccgggcaa gagcaactcg
     2521 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     2581 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     2641 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     2701 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     2761 ccataccaaa cgacgagcgt gacaccacga tgcctgcagc aatggcaaca acgttgcgca
     2821 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     2881 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     2941 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
     3001 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     3061 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     3121 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     3181 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     3241 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     3301 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     3361 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     3421 caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     3481 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     3541 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     3601 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     3661 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     3721 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     3781 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     3841 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     3901 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     3961 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     4021 agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta
     4081 cgcatctgtg cggtatttca caccgcaatg gtgcactctc agtacaatct gctctgatgc
     4141 cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc
     4201 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     4261 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     4321 ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag
     4381 atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt taatgtctgg
     4441 cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac tgatgcctcc
     4501 gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga gaggatgctc
     4561 acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga gggtaaacaa
     4621 ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg ccagcgcttc
     4681 gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat gcagatccgg
     4741 aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac acggaaaccg
     4801 aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt
     4861 cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca gcctagccgg
     4921 gtcctcaacg acaggagcac gatcatgcgc acccgtggcc aggacccaac gctgcccgag
     4981 atgcgccgcg tgcggctgct ggagatggcg gacgcgatgg atatgttctg ccaagggttg
     5041 gtttgcgcat tcacagttct ccgcaagaat tgattggctc caattcttgg agtggtgaat
     5101 ccgttagcga ggtgccgccg gcttccattc aggtcgaggt ggcccggctc catgcaccgc
     5161 gacgcaacgc ggggaggcag acaaggtata gggcggcgcc tacaatccat gccaacccgt
     5221 tccatgtgct cgccgaggcg gcataaatcg ccgtgacgat cagcggtcca atgatcgaag
     5281 ttaggctggt aagagccgcg agcgatcctt gaagctgtcc ctgatggtcg tcatctacct
     5341 gcctggacag catggcctgc aacgcgggca tcccgatgcc gccggaagcg agaagaatca
     5401 taatggggaa ggccatccag cctcgcgtcg cgaacgccag caagacgtag cccagcgcgt
     5461 cggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg gcgggaccag
     5521 tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca
     5581 tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct gccggcacct
     5641 gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata gtcatgcccc
     5701 gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatcccg
     5761 gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt
     5821 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     5881 tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg
     5941 cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc
     6001 aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta
     6061 tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc ggtaatggcg
     6121 cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc
     6181 tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt
     6241 tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga
     6301 cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg
     6361 accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg
     6421 ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca
     6481 gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg
     6541 agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc
     6601 accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc
     6661 gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt
     6721 tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc
     6781 cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag
     6841 acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat
     6901 tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg
     6961 tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag
     7021 gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tg
//
