LOCUS       sequence-178194-        7302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of S. cerevisiae Vps4 AAA ATPase cassette fused
            to P. aeruginosa Hcp1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7302)
  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 7302)
  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)-Hcp1
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     168..187
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     271..290
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        271..289
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    290..314
                     /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             329..351
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             362..379
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             380..421
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             422..442
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(2065..2083)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2079..2126
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(2364..2382)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2450..2554
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2555..2623,2624..3415)
                     /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(2773..2792)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3586..4174
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4075..4094
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4328..4345
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4360..4499
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4425..4444)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4585..4607
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4601..4792)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6104..7186)
                     /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     7148..7167
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7187..7264)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 aggaagcagc ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca
       61 tgcaaggaga tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg
      121 aaacaagcgc tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg
      181 atataggcgc cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg
      241 tagaggatcg agatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag
      301 cggataacaa ttcccctcta gaaataattt tgtttaactt taagaaggag atatacatat
      361 gcatcatcac catcaccatg gtaagcctat ccctaaccct ctcctcggtc tcgattctac
      421 ggaaaacctg tattttcagg gacaggaaga gggtgaggat aatggtggag aagacaacaa
      481 aaagctaaga ggtgccttat cgagtgctat tttgtctgag aagcccaatg ttaagtggga
      541 agacgtggct ggactggaag gagccaaaga ggcccttaaa gaagccgtta ttttacctgt
      601 caagttccca catttgttca aaggtaaccg taagcccact tcgggaatct tattgtacgg
      661 accaccaggt acaggtaaat catatttggc aaaagcagtg gccaccgagg ccaattctac
      721 ctttttcagt gttagttcca gtgatttggt ttctaagtgg atgggtgaat ctgaaaaact
      781 tgtcaaacag ttatttgcga tggcaagaga aaataaacct tctattattt ttatagatga
      841 agtggatgcg ctaacaggta ccagaggaga aggagagagc gaagcaagta gaagaattaa
      901 aacagaatta ttggtacaaa tgaatggtgt tgggaatgac tcccaaggtg ttttggtcct
      961 tggcgcaaca aatatcccat ggcaactgga cagtgccatc aggagaagat ttgaaagaag
     1021 aatatatata ccattaccag atttggcggc aagaaccacc atgttcgaga ttaacgtcgg
     1081 cgacactcca tgcgtattga ccaaggaaga ttatagaact ttaggtgcaa tgactgaagg
     1141 ttattccggg agtgatattg ctgtagtggt gaaagatgcg ctaatgcaac cgataagaaa
     1201 aattcaaagc gcgacacatt tcaaagatgt ctctacggag gacgacgaaa caagaaaact
     1261 aacaccatgc tctccaggtg atgatggcgc catcgagatg agttggacgg atattgaagc
     1321 tgatgaatta aaagaaccag atctcaccat aaaggatttc ttaaaggcta tcaaatcgac
     1381 gagacctacc gtgaatgagg atgacttgct gaagcaagaa cagttcacta gagattttgg
     1441 tcaagaaggt aacggtggtg gcgggtctgg aggtggaggc agtggcggag gtgggagcgg
     1501 aggcgggatg gctgttgata tgttcatcaa gatcggcgac gtcaagggtg agtccaagga
     1561 caagactcac gccgaggaaa tcgacgtgct ggcatggagc tggggcatgt cccagtccgg
     1621 gtcgatgcac atgggcggtg gcggcggcgc cggcaaggtc aacgtgcagg acctgtcgtt
     1681 caccaagtac atcgacaagt ccacgcccaa cctgatgatg gcctgctcca gcggcaagca
     1741 ctatccgcag gcgaagctga ccatccgcaa ggccggcggc gagaaccagg tcgagtacct
     1801 gatcatcacc ctgaaggaag tcctggtgtc ctcggtgagc accggcggca gcggtggcga
     1861 ggatcgcctg accgagaacg tgaccctgaa cttcgcccag gtccaggtcg actaccagcc
     1921 gcagaaggcg gatggcgcga aggacggcgg tccggtcaag tacggctgga acatccgcca
     1981 gaacgtgcag gccggataat aaaagggcga gctcagatcc ggctgctaac aaagcccgaa
     2041 aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct
     2101 ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga tatcccgcaa
     2161 gaggcccggc agtaccggca taaccaagcc tatgcctaca gcatccaggg tgacggtgcc
     2221 gaggatgacg atgagcgcat tgttagattt catacacggt gcctgactgc gttagcaatt
     2281 taactgtgat aaactaccgc attaaagcta gcttatcgat gataagctgt caaacatgag
     2341 aattaattct tgaagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat
     2401 gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     2461 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     2521 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     2581 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     2641 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     2701 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     2761 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt gacgccgggc aagagcaact
     2821 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     2881 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     2941 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     3001 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     3061 agccatacca aacgacgagc gtgacaccac gatgcctgca gcaatggcaa caacgttgcg
     3121 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     3181 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     3241 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc
     3301 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     3361 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     3421 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     3481 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     3541 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     3601 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     3661 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     3721 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     3781 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     3841 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     3901 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     3961 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     4021 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     4081 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     4141 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     4201 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     4261 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     4321 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct
     4381 tacgcatctg tgcggtattt cacaccgcaa tggtgcactc tcagtacaat ctgctctgat
     4441 gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc
     4501 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
     4561 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     4621 caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac
     4681 agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct
     4741 ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct
     4801 ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc
     4861 tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac
     4921 aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct
     4981 tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc
     5041 ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac
     5101 cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg
     5161 ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc
     5221 gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg ccaggaccca acgctgcccg
     5281 agatgcgccg cgtgcggctg ctggagatgg cggacgcgat ggatatgttc tgccaagggt
     5341 tggtttgcgc attcacagtt ctccgcaaga attgattggc tccaattctt ggagtggtga
     5401 atccgttagc gaggtgccgc cggcttccat tcaggtcgag gtggcccggc tccatgcacc
     5461 gcgacgcaac gcggggaggc agacaaggta tagggcggcg cctacaatcc atgccaaccc
     5521 gttccatgtg ctcgccgagg cggcataaat cgccgtgacg atcagcggtc caatgatcga
     5581 agttaggctg gtaagagccg cgagcgatcc ttgaagctgt ccctgatggt cgtcatctac
     5641 ctgcctggac agcatggcct gcaacgcggg catcccgatg ccgccggaag cgagaagaat
     5701 cataatgggg aaggccatcc agcctcgcgt cgcgaacgcc agcaagacgt agcccagcgc
     5761 gtcggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg tggcgggacc
     5821 agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg acaggccgat
     5881 catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg ctgccggcac
     5941 ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc
     6001 ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc
     6061 cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca
     6121 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     6181 tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat
     6241 tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca
     6301 gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg
     6361 tatcgtcgta tcccactacc gagatatccg caccaacgcg cagcccggac tcggtaatgg
     6421 cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc
     6481 cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc
     6541 gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca
     6601 gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg
     6661 cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga
     6721 tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca
     6781 cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg
     6841 cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca
     6901 ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg
     6961 gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca
     7021 gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt
     7081 cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag
     7141 agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc accaccctga
     7201 attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg
     7261 tgtccgggat ctcgacgctc tcccttatgc gactcctgca tt
//
