LOCUS       sequence-175371-        6762 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of a monomeric variant of the S. cerevisiae 
            Vps4 AAA ATPase cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6762)
  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 6762)
  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(101-437) L151D
FEATURES             Location/Qualifiers
     source          1..6762
                     /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(1463..1481)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1477..1524
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1762..1780)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1848..1952
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1953..2021,2022..2813)
                     /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(2171..2190)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2984..3572
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3473..3492
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3726..3743
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3758..3897
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3823..3842)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3983..4005
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3999..4190)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5502..6584)
                     /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     6546..6565
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6585..6662)
                     /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 caggaagagg gtgaggataa tggtggagaa gacaacaaaa
      421 agctaagagg tgccttatcg agtgctattt tgtctgagaa gcccaatgtt aagtgggaag
      481 acgtggctgg actggaagga gccaaagagg cccttaaaga agccgttatt gatcctgtca
      541 agttcccaca tttgttcaaa ggtaaccgta agcccacttc gggaatctta ttgtacggac
      601 caccaggtac aggtaaatca tatttggcaa aagcagtggc caccgaggcc aattctacct
      661 ttttcagtgt tagttccagt gatttggttt ctaagtggat gggtgaatct gaaaaacttg
      721 tcaaacagtt atttgcgatg gcaagagaaa ataaaccttc tattattttt atagatgaag
      781 tggatgcgct aacaggtacc agaggagaag gagagagcga agcaagtaga agaattaaaa
      841 cagaattatt ggtacaaatg aatggtgttg ggaatgactc ccaaggtgtt ttggtccttg
      901 gcgcaacaaa tatcccatgg caactggaca gtgccatcag gagaagattt gaaagaagaa
      961 tatatatacc attaccagat ttggcggcaa gaaccaccat gttcgagatt aacgtcggcg
     1021 acactccatg cgtattgacc aaggaagatt atagaacttt aggtgcaatg actgaaggtt
     1081 attccgggag tgatattgct gtagtggtga aagatgcgct aatgcaaccg ataagaaaaa
     1141 ttcaaagcgc gacacatttc aaagatgtct ctacggagga cgacgaaaca agaaaactaa
     1201 caccatgctc tccaggtgat gatggcgcca tcgagatgag ttggacggat attgaagctg
     1261 atgaattaaa agaaccagat ctcaccataa aggatttctt aaaggctatc aaatcgacga
     1321 gacctaccgt gaatgaggat gacttgctga agcaagaaca gttcactaga gattttggtc
     1381 aagaaggtaa ctagcatatg aagggcgagc tcagatccgg ctgctaacaa agcccgaaag
     1441 gaagctgagt tggctgctgc caccgctgag caataactag cataacccct tggggcctct
     1501 aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggata tcccgcaaga
     1561 ggcccggcag taccggcata accaagccta tgcctacagc atccagggtg acggtgccga
     1621 ggatgacgat gagcgcattg ttagatttca tacacggtgc ctgactgcgt tagcaattta
     1681 actgtgataa actaccgcat taaagctagc ttatcgatga taagctgtca aacatgagaa
     1741 ttaattcttg aagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga
     1801 taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     1861 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     1921 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     1981 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     2041 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     2101 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     2161 ttaaagttct gctatgtggc gcggtattat cccgtgttga cgccgggcaa gagcaactcg
     2221 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     2281 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     2341 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     2401 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     2461 ccataccaaa cgacgagcgt gacaccacga tgcctgcagc aatggcaaca acgttgcgca
     2521 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     2581 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     2641 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
     2701 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     2761 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     2821 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     2881 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     2941 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     3001 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     3061 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     3121 caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     3181 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     3241 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     3301 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     3361 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     3421 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     3481 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     3541 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     3601 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     3661 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     3721 agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta
     3781 cgcatctgtg cggtatttca caccgcaatg gtgcactctc agtacaatct gctctgatgc
     3841 cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc
     3901 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     3961 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     4021 ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag
     4081 atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt taatgtctgg
     4141 cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac tgatgcctcc
     4201 gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga gaggatgctc
     4261 acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga gggtaaacaa
     4321 ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg ccagcgcttc
     4381 gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat gcagatccgg
     4441 aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac acggaaaccg
     4501 aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt
     4561 cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca gcctagccgg
     4621 gtcctcaacg acaggagcac gatcatgcgc acccgtggcc aggacccaac gctgcccgag
     4681 atgcgccgcg tgcggctgct ggagatggcg gacgcgatgg atatgttctg ccaagggttg
     4741 gtttgcgcat tcacagttct ccgcaagaat tgattggctc caattcttgg agtggtgaat
     4801 ccgttagcga ggtgccgccg gcttccattc aggtcgaggt ggcccggctc catgcaccgc
     4861 gacgcaacgc ggggaggcag acaaggtata gggcggcgcc tacaatccat gccaacccgt
     4921 tccatgtgct cgccgaggcg gcataaatcg ccgtgacgat cagcggtcca atgatcgaag
     4981 ttaggctggt aagagccgcg agcgatcctt gaagctgtcc ctgatggtcg tcatctacct
     5041 gcctggacag catggcctgc aacgcgggca tcccgatgcc gccggaagcg agaagaatca
     5101 taatggggaa ggccatccag cctcgcgtcg cgaacgccag caagacgtag cccagcgcgt
     5161 cggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg gcgggaccag
     5221 tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca
     5281 tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct gccggcacct
     5341 gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata gtcatgcccc
     5401 gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatcccg
     5461 gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt
     5521 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     5581 tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg
     5641 cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc
     5701 aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta
     5761 tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc ggtaatggcg
     5821 cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc
     5881 tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt
     5941 tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga
     6001 cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg
     6061 accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg
     6121 ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca
     6181 gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg
     6241 agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc
     6301 accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc
     6361 gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt
     6421 tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc
     6481 cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag
     6541 acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat
     6601 tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg
     6661 tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag
     6721 gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tg
//
