LOCUS       sequence-175359-        7302 bp    DNA     circular SYN 15-APR-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 Apr 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 K321A/R325A
FEATURES             Location/Qualifiers
     source          1..7302
                     /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(2003..2021)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2017..2064
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(2302..2320)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2388..2492
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2493..2561,2562..3353)
                     /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(2711..2730)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3524..4112
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4013..4032
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4266..4283
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4298..4437
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4363..4382)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4523..4545
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4539..4730)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6042..7124)
                     /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     7086..7105
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7125..7202)
                     /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 ttacctgtca
      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 gcggaagatt atgcgacttt 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 cggtggtggc gggtctggag gtggaggcag tggcggaggt gggagcggag
     1441 gcgggatggc tgttgatatg ttcatcaaga tcggcgacgt caagggtgag tccaaggaca
     1501 agactcacgc cgaggaaatc gacgtgctgg catggagctg gggcatgtcc cagtccgggt
     1561 cgatgcacat gggcggtggc ggcggcgccg gcaaggtcaa cgtgcaggac ctgtcgttca
     1621 ccaagtacat cgacaagtcc acgcccaacc tgatgatggc ctgctccagc ggcaagcact
     1681 atccgcaggc gaagctgacc atccgcaagg ccggcggcga gaaccaggtc gagtacctga
     1741 tcatcaccct gaaggaagtc ctggtgtcct cggtgagcac cggcggcagc ggtggcgagg
     1801 atcgcctgac cgagaacgtg accctgaact tcgcccaggt ccaggtcgac taccagccgc
     1861 agaaggcgga tggcgcgaag gacggcggtc cggtcaagta cggctggaac atccgccaga
     1921 acgtgcaggc cggataataa aagggcgagc tcagatccgg ctgctaacaa agcccgaaag
     1981 gaagctgagt tggctgctgc caccgctgag caataactag cataacccct tggggcctct
     2041 aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggata tcccgcaaga
     2101 ggcccggcag taccggcata accaagccta tgcctacagc atccagggtg acggtgccga
     2161 ggatgacgat gagcgcattg ttagatttca tacacggtgc ctgactgcgt tagcaattta
     2221 actgtgataa actaccgcat taaagctagc ttatcgatga taagctgtca aacatgagaa
     2281 ttaattcttg aagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga
     2341 taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     2401 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     2461 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     2521 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     2581 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     2641 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     2701 ttaaagttct gctatgtggc gcggtattat cccgtgttga cgccgggcaa gagcaactcg
     2761 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     2821 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     2881 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     2941 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     3001 ccataccaaa cgacgagcgt gacaccacga tgcctgcagc aatggcaaca acgttgcgca
     3061 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     3121 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     3181 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
     3241 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     3301 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     3361 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     3421 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     3481 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     3541 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     3601 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     3661 caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     3721 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     3781 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     3841 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     3901 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     3961 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     4021 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     4081 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     4141 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     4201 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     4261 agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta
     4321 cgcatctgtg cggtatttca caccgcaatg gtgcactctc agtacaatct gctctgatgc
     4381 cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc
     4441 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     4501 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     4561 ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag
     4621 atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt taatgtctgg
     4681 cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac tgatgcctcc
     4741 gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga gaggatgctc
     4801 acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga gggtaaacaa
     4861 ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg ccagcgcttc
     4921 gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat gcagatccgg
     4981 aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac acggaaaccg
     5041 aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt
     5101 cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca gcctagccgg
     5161 gtcctcaacg acaggagcac gatcatgcgc acccgtggcc aggacccaac gctgcccgag
     5221 atgcgccgcg tgcggctgct ggagatggcg gacgcgatgg atatgttctg ccaagggttg
     5281 gtttgcgcat tcacagttct ccgcaagaat tgattggctc caattcttgg agtggtgaat
     5341 ccgttagcga ggtgccgccg gcttccattc aggtcgaggt ggcccggctc catgcaccgc
     5401 gacgcaacgc ggggaggcag acaaggtata gggcggcgcc tacaatccat gccaacccgt
     5461 tccatgtgct cgccgaggcg gcataaatcg ccgtgacgat cagcggtcca atgatcgaag
     5521 ttaggctggt aagagccgcg agcgatcctt gaagctgtcc ctgatggtcg tcatctacct
     5581 gcctggacag catggcctgc aacgcgggca tcccgatgcc gccggaagcg agaagaatca
     5641 taatggggaa ggccatccag cctcgcgtcg cgaacgccag caagacgtag cccagcgcgt
     5701 cggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg gcgggaccag
     5761 tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca
     5821 tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct gccggcacct
     5881 gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata gtcatgcccc
     5941 gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatcccg
     6001 gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt
     6061 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     6121 tgcgtattgg gcgccagggt ggtttttctc ttcaccagtg agacgggcaa cagctgattg
     6181 cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc
     6241 aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta
     6301 tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc ggtaatggcg
     6361 cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc
     6421 tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt
     6481 tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga
     6541 cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg
     6601 accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg
     6661 ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca
     6721 gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg
     6781 agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc
     6841 accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc
     6901 gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt
     6961 tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc
     7021 cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag
     7081 acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat
     7141 tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg
     7201 tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag
     7261 gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tg
//
