LOCUS       sequence-175364-        5910 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of a VSL domain variant of S. cerevisiae Vta1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5910)
  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 5910)
  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) S278C
FEATURES             Location/Qualifiers
     source          1..5910
                     /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(611..629)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      625..672
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(910..928)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        996..1100
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1101..1169,1170..1961)
                     /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(1319..1338)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2132..2720
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2621..2640
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2874..2891
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2906..3045
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2971..2990)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3131..3153
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3147..3338)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4650..5732)
                     /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     5694..5713
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5733..5810)
                     /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 tgctatacaa aggatgagtt gacaaaaatt atggacaggg
      421 ccagtaaaat agagcaaatt caaaaacttg ccaaatacgc cataagcgca ttgaattatg
      481 aagacctgcc caccgccaaa gatgagttaa ctaaggcgtt ggatctgtta aattctattt
      541 gacatatgaa gggcgagctc agatccggct gctaacaaag cccgaaagga agctgagttg
      601 gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg
      661 aggggttttt tgctgaaagg aggaactata tccggatatc ccgcaagagg cccggcagta
      721 ccggcataac caagcctatg cctacagcat ccagggtgac ggtgccgagg atgacgatga
      781 gcgcattgtt agatttcata cacggtgcct gactgcgtta gcaatttaac tgtgataaac
      841 taccgcatta aagctagctt atcgatgata agctgtcaaa catgagaatt aattcttgaa
      901 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
      961 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     1021 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     1081 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     1141 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     1201 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     1261 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     1321 tatgtggcgc ggtattatcc cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac
     1381 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     1441 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     1501 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     1561 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     1621 acgagcgtga caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg
     1681 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     1741 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     1801 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     1861 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     1921 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     1981 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     2041 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     2101 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     2161 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     2221 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc
     2281 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     2341 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     2401 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     2461 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     2521 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     2581 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     2641 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     2701 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     2761 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     2821 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     2881 cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg
     2941 gtatttcaca ccgcaatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     3001 ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca
     3061 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     3121 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
     3181 aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt
     3241 tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag
     3301 cgggccatgt taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga
     3361 tttctgttca tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt
     3421 actgatgatg aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg
     3481 atgcggcggg accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat
     3541 gtaggtgttc cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg
     3601 cagggcgctg acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat
     3661 gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc
     3721 ggtgattcat tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac
     3781 aggagcacga tcatgcgcac ccgtggccag gacccaacgc tgcccgagat gcgccgcgtg
     3841 cggctgctgg agatggcgga cgcgatggat atgttctgcc aagggttggt ttgcgcattc
     3901 acagttctcc gcaagaattg attggctcca attcttggag tggtgaatcc gttagcgagg
     3961 tgccgccggc ttccattcag gtcgaggtgg cccggctcca tgcaccgcga cgcaacgcgg
     4021 ggaggcagac aaggtatagg gcggcgccta caatccatgc caacccgttc catgtgctcg
     4081 ccgaggcggc ataaatcgcc gtgacgatca gcggtccaat gatcgaagtt aggctggtaa
     4141 gagccgcgag cgatccttga agctgtccct gatggtcgtc atctacctgc ctggacagca
     4201 tggcctgcaa cgcgggcatc ccgatgccgc cggaagcgag aagaatcata atggggaagg
     4261 ccatccagcc tcgcgtcgcg aacgccagca agacgtagcc cagcgcgtcg gccgccatgc
     4321 cggcgataat ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg acgaaggctt
     4381 gagcgagggc gtgcaagatt ccgaataccg caagcgacag gccgatcatc gtcgcgctcc
     4441 agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt cctacgagtt
     4501 gcatgataaa gaagacagtc ataagtgcgg cgacgatagt catgccccgc gcccaccgga
     4561 aggagctgac tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag
     4621 tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     4681 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     4741 gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc
     4801 tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc
     4861 tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc
     4921 actaccgaga tatccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc
     4981 agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt
     5041 tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc
     5101 tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca
     5161 gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc
     5221 acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca
     5281 gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc
     5341 tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc
     5401 accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca
     5461 cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc
     5521 agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg
     5581 cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca
     5641 gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac
     5701 tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc
     5761 gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg
     5821 acgctctccc ttatgcgact cctgcattag gaagcagccc agtagtaggt tgaggccgtt
     5881 gagcaccgcc gccgcaagga atggtgcatg
//
