LOCUS       sequence-358663-        5957 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression for VPS4A MIT domain residues 1-84. Has 
            N-terminal HIS-TAG. Internal ID: WISP05-43.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5957)
  AUTHORS   Wenzel DM, Mackay DR, Skalicky JJ, Paine EL, Miller MS, Ullman KS, 
            Sundquist WI
  TITLE     Comprehensive analysis of the human ESCRT-III-MIT domain interactome
            reveals new cofactors for cytokinetic abscission.
  JOURNAL   Elife. 2022 Sep 15;11:e77779. doi: 10.7554/eLife.77779.
  PUBMED    36107470
REFERENCE   2  (bases 1 to 5957)
  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: "2022-09-15"; volume: "11:e77779. doi"; pages: " 
            10.7554/eLife.77779"
COMMENT     Sequence Label: pET16B VPS4A, residues 1-84
FEATURES             Location/Qualifiers
     source          1..5957
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(10..15,16..32,33..38)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     terminator      complement(213..260)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     256..274
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(583..603)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(610..639)
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     RBS             652..674
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    689..713
                     /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)."
     primer_bind     complement(713..732)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(714..732)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(816..835)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1041..1118
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     complement(1138..1157)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3512..3703
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3697..3719)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3805..3944
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3860..3879
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3959..3976)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4130..4718)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4210..4229)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4889..5680,5681..5749))
                     /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     5512..5531
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5750..5854)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5922..5940
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ttctcatgtt tgacagctta tcatcgataa gctttaatgc ggtagtttat cacagttaaa
       61 ttgctaacgc agtcaggcac cgtgtatgaa atctaacaat gcgctcatcg tcatcctcgg
      121 caccgtcacc ctggatgctg taggcatagg cttggttatg ccggtactgc cgggcctctt
      181 gcgggatatc cggatatagt tcctcctttc agcaaaaaac ccctcaagac ccgtttagag
      241 gccccaaggg gttatgctag ttattgctca gcggtggcag cagccaactc agcttccttt
      301 cgggctttgt tagcagccgg atccttatgg cttcttgccg tgtttctctt tgcttcgtaa
      361 ataatccttc agcttctcgg cccggtctag gtactgcacg cacttggctc gaatgctctc
      421 cttggccttg tcgctgtggg cctcatactt gatagcgtgg aggaagtact ccaccgcatg
      481 ctggtacagc cgcagcgcct cctcgtagtt cttggctttg tcctcctctg tggctttcgt
      541 caccagatca atggctttct gcagggtgct ggtggtcata tggccctgga aatacaggtt
      601 ttcgccgcta tgatgatgat gatgatgatg atgatgatgg cccatggtat atctccttct
      661 taaagttaaa caaaattatt tctagagggg aattgttatc cgctcacaat tcccctatag
      721 tgagtcgtat taatttcgcg ggatcgagat ctcgatcctc tacgccggac gcatcgtggc
      781 cggcatcacc ggcgccacag gtgcggttgc tggcgcctat atcgccgaca tcaccgatgg
      841 ggaagatcgg gctcgccact tcgggctcat gagcgcttgt ttcggcgtgg gtatggtggc
      901 aggccccgtg gccgggggac tgttgggcgc catctccttg catgcaccat tccttgcggc
      961 ggcggtgctc aacggcctca acctactact gggctgcttc ctaatgcagg agtcgcataa
     1021 gggagagcgt cgagatcccg gacaccatcg aatggcgcaa aacctttcgc ggtatggcat
     1081 gatagcgccc ggaagagagt caattcaggg tggtgaatgt gaaaccagta acgttatacg
     1141 atgtcgcaga gtatgccggt gtctcttatc agaccgtttc ccgcgtggtg aaccaggcca
     1201 gccacgtttc tgcgaaaacg cgggaaaaag tggaagcggc gatggcggag ctgaattaca
     1261 ttcccaaccg cgtggcacaa caactggcgg gcaaacagtc gttgctgatt ggcgttgcca
     1321 cctccagtct ggccctgcac gcgccgtcgc aaattgtcgc ggcgattaaa tctcgcgccg
     1381 atcaactggg tgccagcgtg gtggtgtcga tggtagaacg aagcggcgtc gaagcctgta
     1441 aagcggcggt gcacaatctt ctcgcgcaac gcgtcagtgg gctgatcatt aactatccgc
     1501 tggatgacca ggatgccatt gctgtggaag ctgcctgcac taatgttccg gcgttatttc
     1561 ttgatgtctc tgaccagaca cccatcaaca gtattatttt ctcccatgaa gacggtacgc
     1621 gactgggcgt ggagcatctg gtcgcattgg gtcaccagca aatcgcgctg ttagcgggcc
     1681 cattaagttc tgtctcggcg cgtctgcgtc tggctggctg gcataaatat ctcactcgca
     1741 atcaaattca gccgatagcg gaacgggaag gcgactggag tgccatgtcc ggttttcaac
     1801 aaaccatgca aatgctgaat gagggcatcg ttcccactgc gatgctggtt gccaacgatc
     1861 agatggcgct gggcgcaatg cgcgccatta ccgagtccgg gctgcgcgtt ggtgcggata
     1921 tctcggtagt gggatacgac gataccgaag acagctcatg ttatatcccg ccgttaacca
     1981 ccatcaaaca ggattttcgc ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct
     2041 ctcagggcca ggcggtgaag ggcaatcagc tgttgcccgt ctcactggtg aaaagaaaaa
     2101 ccaccctggc gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc
     2161 agctggcacg acagtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtaa
     2221 gttagctcac tcattaggca ccgggatctc gaccgatgcc cttgagagcc ttcaacccag
     2281 tcagctcctt ccggtgggcg cggggcatga ctatcgtcgc cgcacttatg actgtcttct
     2341 ttatcatgca actcgtagga caggtgccgg cagcgctctg ggtcattttc ggcgaggacc
     2401 gctttcgctg gagcgcgacg atgatcggcc tgtcgcttgc ggtattcgga atcttgcacg
     2461 ccctcgctca agccttcgtc actggtcccg ccaccaaacg tttcggcgag aagcaggcca
     2521 ttatcgccgg catggcggcc gacgcgctgg gctacgtctt gctggcgttc gcgacgcgag
     2581 gctggatggc cttccccatt atgattcttc tcgcttccgg cggcatcggg atgcccgcgt
     2641 tgcaggccat gctgtccagg caggtagatg acgaccatca gggacagctt caaggatcgc
     2701 tcgcggctct taccagccta acttcgatca ttggaccgct gatcgtcacg gcgatttatg
     2761 ccgcctcggc gagcacatgg aacgggttgg catggattgt aggcgccgcc ctataccttg
     2821 tctgcctccc cgcgttgcgt cgcggtgcat ggagccgggc cacctcgacc tgaatggaag
     2881 ccggcggcac ctcgctaacg gattcaccac tccaagaatt ggagccaatc aattcttgcg
     2941 gagaactgtg aatgcgcaaa ccaacccttg gcagaacata tccatcgcgt ccgccatctc
     3001 cagcagccgc acgcggcgca tctcgggcag cgttgggtcc tggccacggg tgcgcatgat
     3061 cgtgctcctg tcgttgagga cccggctagg ctggcggggt tgccttactg gttagcagaa
     3121 tgaatcaccg atacgcgagc gaacgtgaag cgactgctgc tgcaaaacgt ctgcgacctg
     3181 agcaacaaca tgaatggtct tcggtttccg tgtttcgtaa agtctggaaa cgcggaagtc
     3241 agcgccctgc accattatgt tccggatctg catcgcagga tgctgctggc taccctgtgg
     3301 aacacctaca tctgtattaa cgaagcgctg gcattgaccc tgagtgattt ttctctggtc
     3361 ccgccgcatc cataccgcca gttgtttacc ctcacaacgt tccagtaacc gggcatgttc
     3421 atcatcagta acccgtatcg tgagcatcct ctctcgtttc atcggtatca ttacccccat
     3481 gaacagaaat cccccttaca cggaggcatc agtgaccaaa caggaaaaaa ccgcccttaa
     3541 catggcccgc tttatcagaa gccagacatt aacgcttctg gagaaactca acgagctgga
     3601 cgcggatgaa caggcagaca tctgtgaatc gcttcacgac cacgctgatg agctttaccg
     3661 cagctgcctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga
     3721 gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc
     3781 agcgggtgtt ggcgggtgtc ggggcgcagc catgacccag tcacgtagcg atagcggagt
     3841 gtatactggc ttaactatgc ggcatcagag cagattgtac tgagagtgca ccattgcggt
     3901 gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggcgctct tccgcttcct
     3961 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     4021 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     4081 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     4141 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     4201 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     4261 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     4321 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     4381 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     4441 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     4501 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     4561 acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     4621 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     4681 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     4741 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     4801 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     4861 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     4921 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     4981 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     5041 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     5101 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     5161 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctgcaggc atcgtggtgt
     5221 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     5281 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     5341 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     5401 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     5461 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaacacgg gataataccg
     5521 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     5581 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     5641 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     5701 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     5761 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     5821 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     5881 acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc
     5941 cctttcgtct tcaagaa
//
