LOCUS       sequence-358667-        5255 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression for VPS4B MIT domain, residues 1-86. Has 
            N-terminal GST tag. Internal ID: WISP04-155..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5255)
  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 5255)
  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: pGEX2T VPS4B, residues 1-86
FEATURES             Location/Qualifiers
     source          1..5255
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(181..186,187..202,203..209)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    217..233
                     /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)."
     CDS             256..909
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     867..889
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             916..933
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             937..957
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(1303..1325)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1470..1488)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1556..1660
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1661..1729,1730..2521)
                     /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(1879..1898)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2692..3280
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3181..3200
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3434..3451
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3524..3601
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3602..4684
                     /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     complement(3621..3640)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(4733..4738,4739..4756,4757..4763)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4771..4787
                     /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     4795..4811
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4795..4811
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(4807..5255,1..79)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(4827..4844)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4827..4843)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4836..4858)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 cttatcgact gcacggtgca ccaatgcttc tggcgtcagg cagccatcgg aagctgtggt
       61 atggctgtgc aggtcgtaaa tcactgcata attcgtgtcg ctcaaggcgc actcccgttc
      121 tggataatgt tttttgcgcc gacatcataa cggttctggc aaatattctg aaatgagctg
      181 ttgacaatta atcatcggct cgtataatgt gtggaattgt gagcggataa caatttcaca
      241 caggaaacag tattcatgtc ccctatacta ggttattgga aaattaaggg ccttgtgcaa
      301 cccactcgac ttcttttgga atatcttgaa gaaaaatatg aagagcattt gtatgagcgc
      361 gatgaaggtg ataaatggcg aaacaaaaag tttgaattgg gtttggagtt tcccaatctt
      421 ccttattata ttgatggtga tgttaaatta acacagtcta tggccatcat acgttatata
      481 gctgacaagc acaacatgtt gggtggttgt ccaaaagagc gtgcagagat ttcaatgctt
      541 gaaggagcgg ttttggatat tagatacggt gtttcgagaa ttgcatatag taaagacttt
      601 gaaactctca aagttgattt tcttagcaag ctacctgaaa tgctgaaaat gttcgaagat
      661 cgtttatgtc ataaaacata tttaaatggt gatcatgtaa cccatcctga cttcatgttg
      721 tatgacgctc ttgatgttgt tttatacatg gacccaatgt gcctggatgc gttcccaaaa
      781 ttagtttgtt ttaaaaaacg tattgaagct atcccacaaa ttgataagta cttgaaatcc
      841 agcaagtata tagcatggcc tttgcagggc tggcaagcca cgtttggtgg tggcgaccat
      901 cctccaaaat cggatctggt tccgcgtgga tctccggaaa acctgtattt ccagggccat
      961 atgatgtcat ccacttcgcc caacctccag aaagcgatag atctggctag caaagcagcg
     1021 caagaagaca aggctgggaa ctacgaagaa gcccttcagc tctatcagca tgctgtgcag
     1081 tattttcttc atgtcgttaa atatgaagca cagggtgata aagccaagca aagtatcagg
     1141 gcaaagtgta cagaatatct tgatagagca gaaaaactaa aggagtacct gaaaaataaa
     1201 gagaaaaaag cacagaagcc ataaggatcc tcgagaattc tcgagaattc atcgtgactg
     1261 actgacgatc tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct
     1321 cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg
     1381 cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac gtagcgatag
     1441 cggagtgtat aattcttgaa gacgaaaggg cctcgtgata cgcctatttt tataggttaa
     1501 tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg
     1561 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     1621 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     1681 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     1741 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     1801 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     1861 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtgttgacg ccgggcaaga
     1921 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     1981 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     2041 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     2101 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     2161 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgcagcaa tggcaacaac
     2221 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     2281 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     2341 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
     2401 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     2461 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     2521 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     2581 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     2641 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     2701 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     2761 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     2821 gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     2881 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     2941 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     3001 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     3061 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     3121 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     3181 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     3241 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     3301 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     3361 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     3421 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt
     3481 tctccttacg catctgtgcg gtatttcaca ccgcataaat tccgacacca tcgaatggcg
     3541 caaaaccttt cgcggtatgg catgatagcg cccggaagag agtcaattca gggtggtgaa
     3601 tgtgaaacca gtaacgttat acgatgtcgc agagtatgcc ggtgtctctt atcagaccgt
     3661 ttcccgcgtg gtgaaccagg ccagccacgt ttctgcgaaa acgcgggaaa aagtggaagc
     3721 ggcgatggcg gagctgaatt acattcccaa ccgcgtggca caacaactgg cgggcaaaca
     3781 gtcgttgctg attggcgttg ccacctccag tctggccctg cacgcgccgt cgcaaattgt
     3841 cgcggcgatt aaatctcgcg ccgatcaact gggtgccagc gtggtggtgt cgatggtaga
     3901 acgaagcggc gtcgaagcct gtaaagcggc ggtgcacaat cttctcgcgc aacgcgtcag
     3961 tgggctgatc attaactatc cgctggatga ccaggatgcc attgctgtgg aagctgcctg
     4021 cactaatgtt ccggcgttat ttcttgatgt ctctgaccag acacccatca acagtattat
     4081 tttctcccat gaagacggta cgcgactggg cgtggagcat ctggtcgcat tgggtcacca
     4141 gcaaatcgcg ctgttagcgg gcccattaag ttctgtctcg gcgcgtctgc gtctggctgg
     4201 ctggcataaa tatctcactc gcaatcaaat tcagccgata gcggaacggg aaggcgactg
     4261 gagtgccatg tccggttttc aacaaaccat gcaaatgctg aatgagggca tcgttcccac
     4321 tgcgatgctg gttgccaacg atcagatggc gctgggcgca atgcgcgcca ttaccgagtc
     4381 cgggctgcgc gttggtgcgg atatctcggt agtgggatac gacgataccg aagacagctc
     4441 atgttatatc ccgccgttaa ccaccatcaa acaggatttt cgcctgctgg ggcaaaccag
     4501 cgtggaccgc ttgctgcaac tctctcaggg ccaggcggtg aagggcaatc agctgttgcc
     4561 cgtctcactg gtgaaaagaa aaaccaccct ggcgcccaat acgcaaaccg cctctccccg
     4621 cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca
     4681 gtgagcgcaa cgcaattaat gtaagttagc tcactcatta ggcaccccag gctttacact
     4741 ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa
     4801 acagctatga ccatgattac ggattcactg gccgtcgttt tacaacgtcg tgactgggaa
     4861 aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt
     4921 aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa
     4981 tggcgctttg cctggtttcc ggcaccagaa gcggtgccgg aaagctggct ggagtgcgat
     5041 cttcctgagg ccgatactgt cgtcgtcccc tcaaactggc agatgcacgg ttacgatgcg
     5101 cccatctaca ccaacgtgac ctatcccatt acggtcaatc cgccgtttgt tcccacggag
     5161 aatccgacgg gttgttactc gctcacattt aatgttgatg aaagctggct acaggaaggc
     5221 cagacgcgaa ttatttttga tggcgttgga attag
//
