LOCUS       sequence-365338-        6125 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of VPS4B (S2C mutation).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6125)
  AUTHORS   Cada AK, Pavlin MR, Castillo JP, Tong AB, Larsen KP, Ren X, Yokom 
            AL, Tsai FC, Shiah JV, Bassereau PM, Bustamante CJ, Hurley JH
  TITLE     Friction-driven membrane scission by the human ESCRT-III proteins 
            CHMP1B and IST1.
  JOURNAL   Proc Natl Acad Sci U S A. 2022 Jul 19;119(29):e2204536119. doi: 
            10.1073/pnas.2204536119. Epub 2022 Jul 11.
  PUBMED    35858336
REFERENCE   2  (bases 1 to 6125)
  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: "Proc Natl 
            Acad Sci U S A."; date: "2022-07-19"; volume: "119(29):e2204536119. 
            doi"; pages: " 10.1073/pnas.2204536119. Epub 2022 Jul 11"
COMMENT     Sequence Label: VPS4B, S2C
FEATURES             Location/Qualifiers
     source          1..6125
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     43..62
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     296..313
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    328..467
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(393..412)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     553..575
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(569..760)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     2218..2237
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2321..2340
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2321..2339
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             2428..2450
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2470..2487
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2488..2508
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             3338..3349
                     /codon_start=1
                     /product="WELQut protease recognition and cleavage site"
                     /label=WELQut site
                     /translation="WELQ"
     primer_bind     complement(3975..3993)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3989..4036
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(4402..4407,4408..4424,4425..4430))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(4457..4475)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4543..4647
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4648..4716,4717..5508)
                     /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(4866..4885)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(5679..6125,1..142)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
       61 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
      121 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
      181 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
      241 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
      301 gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg
      361 cggtatttca caccgcaatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
      421 agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc
      481 caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag
      541 ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg
      601 cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct
      661 gttcatccgc gtccagctcg ttgagtttct ccagaagcgt taatgtctgg cttctgataa
      721 agcgggccat gttaagggcg gttttttcct gtttggtcac tgatgcctcc gtgtaagggg
      781 gatttctgtt catgggggta atgataccga tgaaacgaga gaggatgctc acgatacggg
      841 ttactgatga tgaacatgcc cggttactgg aacgttgtga gggtaaacaa ctggcggtat
      901 ggatgcggcg ggaccagaga aaaatcactc agggtcaatg ccagcgcttc gttaatacag
      961 atgtaggtgt tccacagggt agccagcagc atcctgcgat gcagatccgg aacataatgg
     1021 tgcagggcgc tgacttccgc gtttccagac tttacgaaac acggaaaccg aagaccattc
     1081 atgttgttgc tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta
     1141 tcggtgattc attctgctaa ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg
     1201 acaggagcac gatcatgcgc acccgtggcc aggacccaac gctgcccgag atgcgccgcg
     1261 tgcggctgct ggagatggcg gacgcgatgg atatgttctg ccaagggttg gtttgcgcat
     1321 tcacagttct ccgcaagaat tgattggctc caattcttgg agtggtgaat ccgttagcga
     1381 ggtgccgccg gcttccattc aggtcgaggt ggcccggctc catgcaccgc gacgcaacgc
     1441 ggggaggcag acaaggtata gggcggcgcc tacaatccat gccaacccgt tccatgtgct
     1501 cgccgaggcg gcataaatcg ccgtgacgat cagcggtcca atgatcgaag ttaggctggt
     1561 aagagccgcg agcgatcctt gaagctgtcc ctgatggtcg tcatctacct gcctggacag
     1621 catggcctgc aacgcgggca tcccgatgcc gccggaagcg agaagaatca taatggggaa
     1681 ggccatccag cctcgcgtcg cgaacgccag caagacgtag cccagcgcgt cggccgccat
     1741 gccggcgata atggcctgct tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc
     1801 ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct
     1861 ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct gccggcacct gtcctacgag
     1921 ttgcatgata aagaagacag tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg
     1981 gaaggagctg actgggttga aggctctcaa gggcatcggt cgacgctctc ccttatgcga
     2041 ctcctgcatt aggaagcagc ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag
     2101 gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg gccacggggc ctgccaccat
     2161 acccacgccg aaacaagcgc tcatgagccc gaagtggcga gcccgatctt ccccatcggt
     2221 gatgtcggcg atataggcgc cagcaaccgc acctgtggcg ccggtgatgc cggccacgat
     2281 gcgtccggcg tagaggatcg agatctcgat cccgcgaaat taatacgact cactataggg
     2341 agaccacaac ggtttccctc tagtgccggc tccggagagc tctttaatta agcggccgcc
     2401 ctgcaggact cgagttctag aaataatttt gtttaacttt aagaaggaga tatacatatg
     2461 aaatcttctc accatcacca tcaccatgaa aacctgtact tccaatccaa tgcaatgtgc
     2521 tccacttcgc ccaacctcca gaaagcgata gatctggcta gcaaagcagc gcaagaagac
     2581 aaggctggga actacgaaga agcccttcag ctctatcagc atgctgtgca gtattttctt
     2641 catgtcgtta aatatgaagc acagggtgat aaagccaagc aaagtatcag ggcaaagtgt
     2701 acagaatatc ttgatagagc agaaaaacta aaggagtacc tgaaaaataa agagaaaaaa
     2761 gcacagaagc cagtgaaaga aggacagccg agtccagcag atgagaaggg gaatgacagt
     2821 gatggggaag gagaatctga tgatcctgaa aaaaagaaac tacagaatca acttcaaggt
     2881 gccattgtta tagaacgacc aaatgtgaaa tggagtgacg ttgctggact tgaaggagcc
     2941 aaagaagcac tgaaagaggc tgtgatactg cctattaaat ttcctcatct ttttacaggc
     3001 aagagaacac cttggagggg aatcctatta tttgggccgc ctggaacagg aaagtcctac
     3061 ttagccaaag ctgtagcaac agaagccaac aactcaacat ttttttcaat atcttcctct
     3121 gatcttgttt ctaagtggct aggtgaaagt gaaaaactgg ttaagaattt attccaactt
     3181 gccagagaga acaagccctc cattatcttc attgatgaaa ttgattctct ctgtggttca
     3241 agaagtgaaa atgaaagtga agccgcacgt agaattaaga cggagttcct agtgcaaatg
     3301 caaggggttg gtgtagacaa tgatggaatt ttggttctgg gagctacaaa tataccctgg
     3361 gttctggatt ctgccattag gcgaagattt gagaaacgaa tttatattcc cttgccggaa
     3421 ccccatgccc gagcagcaat gtttaaactg cacctaggga ccactcagaa cagtctcacg
     3481 gaagcagact ttcgggaact tgggaggaaa acagatggtt attcaggggc agatataagt
     3541 atcattgtac gtgatgccct tatgcagcct gttaggaaag tacagtcagc tactcatttt
     3601 aaaaaggttc gcggaccttc ccgagctgat cctaaccatc ttgtagatga tctgctaaca
     3661 ccttgctctc caggtgaccc tggtgccatt gaaatgacat ggatggatgt ccctggagat
     3721 aaacttttgg agccagttgt ttccatgtcg gatatgttgc ggtcactatc taacacaaaa
     3781 cctacagtca atgaacatga cttgttgaaa ttaaagaagt ttacagaaga ttttggtcaa
     3841 gaaggctaat aacattggaa gtggataacg gatccgcgat cgcggcgcgc cacctggtgg
     3901 ccggccggta ccacgcgtgc gcgctgatcc ggctgctaac aaagcccgaa aggaagctga
     3961 gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt
     4021 cttgaggggt tttttgctga aaggaggaac tatatccgga tatccacagg acgggtgtgg
     4081 tcgccatgat cgcgtagtcg atagtggctc caagtagcga agcgagcagg actgggcggc
     4141 ggccaaagcg gtcggacagt gctccgagaa cgggtgcgca tagaaattgc atcaacgcat
     4201 atagcgctag cagcacgcca tagtgactgg cgatgctgtc ggaatggacg atatcccgca
     4261 agaggcccgg cagtaccggc ataaccaagc ctatgcctac agcatccagg gtgacggtgc
     4321 cgaggatgac gatgagcgca ttgttagatt tcatacacgg tgcctgactg cgttagcaat
     4381 ttaactgtga taaactaccg cattaaagct tatcgatgat aagctgtcaa acatgagaat
     4441 tcttgaagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata
     4501 atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     4561 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     4621 cttcaataac attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     4681 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     4741 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     4801 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     4861 gttctgctat gtggcgcggt attatcccgt gttgacgccg ggcaagagca actcggtcgc
     4921 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     4981 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     5041 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     5101 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     5161 ccaaacgacg agcgtgacac cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta
     5221 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     5281 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     5341 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
     5401 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     5461 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     5521 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     5581 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     5641 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     5701 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     5761 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     5821 actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     5881 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     5941 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     6001 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     6061 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     6121 gtaag
//
