LOCUS       sequence-358576-        5361 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression for VPS9D1 MIT domain, residues 1-97. Has 
            N-terminal HIS-GST tag. Internal ID: WISP20-89..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5361)
  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 5361)
  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: pGEX-THTAP-VPS9D1, residues 1-97
FEATURES             Location/Qualifiers
     source          1..5361
                     /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..954
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             955..975
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             976..1020
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             1021..1044
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(1409..1431)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1576..1594)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1662..1766
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1767..1835,1836..2627)
                     /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(1985..2004)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2798..3386
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3287..3306
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3540..3557
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3630..3707
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3708..4790
                     /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(3727..3746)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(4839..4844,4845..4862,4863..4869)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4877..4893
                     /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     4901..4917
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4901..4917
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(4913..5361,1..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(4933..4950)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4933..4949)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4942..4964)
                     /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 tctccgcacc atcaccatca ccatgaaaac
      961 ctgtattttc agggcggcct gaacgatatt tttgaagcgc agaaaattga atggcatgaa
     1021 cttgaagtcc tctttcaggg acccatggcc gctgcggccg gggacggcac ggtgaagccg
     1081 ctgcagagcg ccatgaagct tgccaacggg gccatcgagc tggacaccgg caaccggccc
     1141 cgggaggcat acacggaata cctgaggagc atccactata tctcccaggt gttactagaa
     1201 gaagtggaaa ccactaaaga agctggggaa actgtgcccc ccgacacctc caagatgctg
     1261 aagctagcac agcagtgtct ggagagggcc cagtcgacgg ccgccaagct tgggaaaaca
     1321 cgcctgaagc caacctgaca ggatcctcga gaattcatcg tgactgactg acgatctgcc
     1381 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
     1441 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
     1501 ttggcgggtg tcggggcgca gccatgaccc agtcacgtag cgatagcgga gtgtataatt
     1561 cttgaagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa
     1621 tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     1681 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     1741 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     1801 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     1861 aagatgctga agatcagttg ggtgcacgag taggttacat cgaactggat ctcaacagcg
     1921 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     1981 ttctgctatg tggcgcggta ttatcccgtg ttgacgccgg gcaagagcaa ctcggtcgcc
     2041 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     2101 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     2161 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     2221 acatgggaga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     2281 caaacgacga gcgtgacacc acgatgcctg cagcaatggc aacaacgttg cgcaaactat
     2341 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     2401 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     2461 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
     2521 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     2581 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     2641 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     2701 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     2761 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     2821 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     2881 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     2941 ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     3001 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     3061 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     3121 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     3181 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     3241 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     3301 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     3361 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     3421 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     3481 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     3541 gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc
     3601 tgtgcggtat ttcacaccgc ataaattccg acaccatcga atggcgcaaa acctttcgcg
     3661 gtatggcatg atagcgcccg gaagagagtc aattcagggt ggtgaatgtg aaaccagtaa
     3721 cgttatacga tgtcgcagag tatgccggtg tctcttatca gaccgtttcc cgcgtggtga
     3781 accaggccag ccacgtttct gcgaaaacgc gggaaaaagt ggaagcggcg atggcggagc
     3841 tgaattacat tcccaaccgc gtggcacaac aactggcggg caaacagtcg ttgctgattg
     3901 gcgttgccac ctccagtctg gccctgcacg cgccgtcgca aattgtcgcg gcgattaaat
     3961 ctcgcgccga tcaactgggt gccagcgtgg tggtgtcgat ggtagaacga agcggcgtcg
     4021 aagcctgtaa agcggcggtg cacaatcttc tcgcgcaacg cgtcagtggg ctgatcatta
     4081 actatccgct ggatgaccag gatgccattg ctgtggaagc tgcctgcact aatgttccgg
     4141 cgttatttct tgatgtctct gaccagacac ccatcaacag tattattttc tcccatgaag
     4201 acggtacgcg actgggcgtg gagcatctgg tcgcattggg tcaccagcaa atcgcgctgt
     4261 tagcgggccc attaagttct gtctcggcgc gtctgcgtct ggctggctgg cataaatatc
     4321 tcactcgcaa tcaaattcag ccgatagcgg aacgggaagg cgactggagt gccatgtccg
     4381 gttttcaaca aaccatgcaa atgctgaatg agggcatcgt tcccactgcg atgctggttg
     4441 ccaacgatca gatggcgctg ggcgcaatgc gcgccattac cgagtccggg ctgcgcgttg
     4501 gtgcggatat ctcggtagtg ggatacgacg ataccgaaga cagctcatgt tatatcccgc
     4561 cgttaaccac catcaaacag gattttcgcc tgctggggca aaccagcgtg gaccgcttgc
     4621 tgcaactctc tcagggccag gcggtgaagg gcaatcagct gttgcccgtc tcactggtga
     4681 aaagaaaaac caccctggcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt
     4741 cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca
     4801 attaatgtaa gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct
     4861 cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat
     4921 gattacggat tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac
     4981 ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc
     5041 ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggc gctttgcctg
     5101 gtttccggca ccagaagcgg tgccggaaag ctggctggag tgcgatcttc ctgaggccga
     5161 tactgtcgtc gtcccctcaa actggcagat gcacggttac gatgcgccca tctacaccaa
     5221 cgtgacctat cccattacgg tcaatccgcc gtttgttccc acggagaatc cgacgggttg
     5281 ttactcgctc acatttaatg ttgatgaaag ctggctacag gaaggccaga cgcgaattat
     5341 ttttgatggc gttggaatta g
//
