LOCUS       sequence-175361-        5904 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of the VSL domain of S. cerevisiae Vta1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5904)
  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 5904)
  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-Vta1(280-330)
FEATURES             Location/Qualifiers
     source          1..5904
                     /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(605..623)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      619..666
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(904..922)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        990..1094
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1095..1163,1164..1955)
                     /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(1313..1332)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2126..2714
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2615..2634
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2868..2885
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2900..3039
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2965..2984)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3125..3147
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3141..3332)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4644..5726)
                     /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     5688..5707
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5727..5804)
                     /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 acaaaggatg agttgacaaa aattatggac agggccagta
      421 aaatagagca aattcaaaaa cttgccaaat acgccataag cgcattgaat tatgaagacc
      481 tgcccaccgc caaagatgag ttaactaagg cgttggatct gttaaattct atttgacata
      541 tgaagggcga gctcagatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct
      601 gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt
      661 tttttgctga aaggaggaac tatatccgga tatcccgcaa gaggcccggc agtaccggca
      721 taaccaagcc tatgcctaca gcatccaggg tgacggtgcc gaggatgacg atgagcgcat
      781 tgttagattt catacacggt gcctgactgc gttagcaatt taactgtgat aaactaccgc
      841 attaaagcta gcttatcgat gataagctgt caaacatgag aattaattct tgaagacgaa
      901 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga
      961 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     1021 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     1081 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     1141 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     1201 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     1261 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     1321 gcgcggtatt atcccgtgtt gacgccgggc aagagcaact cggtcgccgc atacactatt
     1381 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     1441 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     1501 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     1561 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     1621 gtgacaccac gatgcctgca gcaatggcaa caacgttgcg caaactatta actggcgaac
     1681 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     1741 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     1801 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     1861 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     1921 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     1981 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     2041 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     2101 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     2161 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     2221 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag
     2281 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     2341 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     2401 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     2461 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     2521 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     2581 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     2641 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     2701 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     2761 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     2821 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     2881 gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt
     2941 cacaccgcaa tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta
     3001 tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc
     3061 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
     3121 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag
     3181 ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac agatgtctgc ctgttcatcc
     3241 gcgtccagct cgttgagttt ctccagaagc gttaatgtct ggcttctgat aaagcgggcc
     3301 atgttaaggg cggttttttc ctgtttggtc actgatgcct ccgtgtaagg gggatttctg
     3361 ttcatggggg taatgatacc gatgaaacga gagaggatgc tcacgatacg ggttactgat
     3421 gatgaacatg cccggttact ggaacgttgt gagggtaaac aactggcggt atggatgcgg
     3481 cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac agatgtaggt
     3541 gttccacagg gtagccagca gcatcctgcg atgcagatcc ggaacataat ggtgcagggc
     3601 gctgacttcc gcgtttccag actttacgaa acacggaaac cgaagaccat tcatgttgtt
     3661 gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat
     3721 tcattctgct aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc
     3781 acgatcatgc gcacccgtgg ccaggaccca acgctgcccg agatgcgccg cgtgcggctg
     3841 ctggagatgg cggacgcgat ggatatgttc tgccaagggt tggtttgcgc attcacagtt
     3901 ctccgcaaga attgattggc tccaattctt ggagtggtga atccgttagc gaggtgccgc
     3961 cggcttccat tcaggtcgag gtggcccggc tccatgcacc gcgacgcaac gcggggaggc
     4021 agacaaggta tagggcggcg cctacaatcc atgccaaccc gttccatgtg ctcgccgagg
     4081 cggcataaat cgccgtgacg atcagcggtc caatgatcga agttaggctg gtaagagccg
     4141 cgagcgatcc ttgaagctgt ccctgatggt cgtcatctac ctgcctggac agcatggcct
     4201 gcaacgcggg catcccgatg ccgccggaag cgagaagaat cataatgggg aaggccatcc
     4261 agcctcgcgt cgcgaacgcc agcaagacgt agcccagcgc gtcggccgcc atgccggcga
     4321 taatggcctg cttctcgccg aaacgtttgg tggcgggacc agtgacgaag gcttgagcga
     4381 gggcgtgcaa gattccgaat accgcaagcg acaggccgat catcgtcgcg ctccagcgaa
     4441 agcggtcctc gccgaaaatg acccagagcg ctgccggcac ctgtcctacg agttgcatga
     4501 taaagaagac agtcataagt gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc
     4561 tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct
     4621 aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     4681 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg
     4741 gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac cgcctggccc
     4801 tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg
     4861 atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta tcccactacc
     4921 gagatatccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc
     4981 atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag catttgcatg
     5041 gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt
     5101 tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt
     5161 aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc
     5221 agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg gtcagagaca
     5281 tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc atcctggtca
     5341 tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc
     5401 gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct ggcacccagt
     5461 tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg
     5521 gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc cacgcggttg
     5581 ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt cgcagaaacg
     5641 tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc atactctgcg
     5701 acatcgtata acgttactgg tttcacattc accaccctga attgactctc ttccgggcgc
     5761 tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc
     5821 tcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc cgttgagcac
     5881 cgccgccgca aggaatggtg catg
//
