LOCUS       sequence-175366-        6822 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of N-terminally truncated S. cerevisiae Vps4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6822)
  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 6822)
  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(81-437)
FEATURES             Location/Qualifiers
     source          1..6822
                     /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(1523..1541)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1537..1584
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1822..1840)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1908..2012
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2013..2081,2082..2873)
                     /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(2231..2250)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3044..3632
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3533..3552
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3786..3803
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3818..3957
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3883..3902)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4043..4065
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4059..4250)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5562..6644)
                     /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
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQDQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     6606..6625
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6645..6722)
                     /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 gcggcaaaga aatctcctag cgctggtagt ggttccaacg
      421 gtggtaataa gaaaatctct caggaagagg gtgaggataa tggtggagaa gacaacaaaa
      481 agctaagagg tgccttatcg agtgctattt tgtctgagaa gcccaatgtt aagtgggaag
      541 acgtggctgg actggaagga gccaaagagg cccttaaaga agccgttatt ttacctgtca
      601 agttcccaca tttgttcaaa ggtaaccgta agcccacttc gggaatctta ttgtacggac
      661 caccaggtac aggtaaatca tatttggcaa aagcagtggc caccgaggcc aattctacct
      721 ttttcagtgt tagttccagt gatttggttt ctaagtggat gggtgaatct gaaaaacttg
      781 tcaaacagtt atttgcgatg gcaagagaaa ataaaccttc tattattttt atagatgaag
      841 tggatgcgct aacaggtacc agaggagaag gagagagcga agcaagtaga agaattaaaa
      901 cagaattatt ggtacaaatg aatggtgttg ggaatgactc ccaaggtgtt ttggtccttg
      961 gcgcaacaaa tatcccatgg caactggaca gtgccatcag gagaagattt gaaagaagaa
     1021 tatatatacc attaccagat ttggcggcaa gaaccaccat gttcgagatt aacgtcggcg
     1081 acactccatg cgtattgacc aaggaagatt atagaacttt aggtgcaatg actgaaggtt
     1141 attccgggag tgatattgct gtagtggtga aagatgcgct aatgcaaccg ataagaaaaa
     1201 ttcaaagcgc gacacatttc aaagatgtct ctacggagga cgacgaaaca agaaaactaa
     1261 caccatgctc tccaggtgat gatggcgcca tcgagatgag ttggacggat attgaagctg
     1321 atgaattaaa agaaccagat ctcaccataa aggatttctt aaaggctatc aaatcgacga
     1381 gacctaccgt gaatgaggat gacttgctga agcaagaaca gttcactaga gattttggtc
     1441 aagaaggtaa ctagcatatg aagggcgagc tcagatccgg ctgctaacaa agcccgaaag
     1501 gaagctgagt tggctgctgc caccgctgag caataactag cataacccct tggggcctct
     1561 aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggata tcccgcaaga
     1621 ggcccggcag taccggcata accaagccta tgcctacagc atccagggtg acggtgccga
     1681 ggatgacgat gagcgcattg ttagatttca tacacggtgc ctgactgcgt tagcaattta
     1741 actgtgataa actaccgcat taaagctagc ttatcgatga taagctgtca aacatgagaa
     1801 ttaattcttg aagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga
     1861 taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     1921 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     1981 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     2041 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     2101 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     2161 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     2221 ttaaagttct gctatgtggc gcggtattat cccgtgttga cgccgggcaa gagcaactcg
     2281 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     2341 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     2401 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     2461 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     2521 ccataccaaa cgacgagcgt gacaccacga tgcctgcagc aatggcaaca acgttgcgca
     2581 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     2641 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     2701 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
     2761 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     2821 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     2881 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     2941 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     3001 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     3061 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     3121 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     3181 caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     3241 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     3301 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     3361 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     3421 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     3481 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     3541 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     3601 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     3661 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     3721 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     3781 agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta
     3841 cgcatctgtg cggtatttca caccgcaatg gtgcactctc agtacaatct gctctgatgc
     3901 cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc
     3961 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     4021 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     4081 ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag
     4141 atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt taatgtctgg
     4201 cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac tgatgcctcc
     4261 gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga gaggatgctc
     4321 acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga gggtaaacaa
     4381 ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg ccagcgcttc
     4441 gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat gcagatccgg
     4501 aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac acggaaaccg
     4561 aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt
     4621 cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca gcctagccgg
     4681 gtcctcaacg acaggagcac gatcatgcgc acccgtggcc aggacccaac gctgcccgag
     4741 atgcgccgcg tgcggctgct ggagatggcg gacgcgatgg atatgttctg ccaagggttg
     4801 gtttgcgcat tcacagttct ccgcaagaat tgattggctc caattcttgg agtggtgaat
     4861 ccgttagcga ggtgccgccg gcttccattc aggtcgaggt ggcccggctc catgcaccgc
     4921 gacgcaacgc ggggaggcag acaaggtata gggcggcgcc tacaatccat gccaacccgt
     4981 tccatgtgct cgccgaggcg gcataaatcg ccgtgacgat cagcggtcca atgatcgaag
     5041 ttaggctggt aagagccgcg agcgatcctt gaagctgtcc ctgatggtcg tcatctacct
     5101 gcctggacag catggcctgc aacgcgggca tcccgatgcc gccggaagcg agaagaatca
     5161 taatggggaa ggccatccag cctcgcgtcg cgaacgccag caagacgtag cccagcgcgt
     5221 cggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg gcgggaccag
     5281 tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca
     5341 tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct gccggcacct
     5401 gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata gtcatgcccc
     5461 gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatcccg
     5521 gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt
     5581 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     5641 tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg
     5701 cccttcaccg cctggtcctg agagagttgc agcaagcggt ccacgctggt ttgccccagc
     5761 aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta
     5821 tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc ggtaatggcg
     5881 cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc
     5941 tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt
     6001 tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga
     6061 cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg
     6121 accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg
     6181 ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca
     6241 gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg
     6301 agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc
     6361 accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc
     6421 gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt
     6481 tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc
     6541 cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag
     6601 acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat
     6661 tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg
     6721 tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag
     6781 gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tg
//
