LOCUS       sequence-245275-        5039 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses residues 186-199 with L to A mutations at residues 192 and
            195 of CHMP1B with an N-terminal GST tag and a TEV cleavage site; 
            Internal ID: WISP 09-287.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5039)
  AUTHORS   Nguyen HC, Talledge N, McCullough J, Sharma A, Moss FR 3rd, Iwasa 
            JH, Vershinin MD, Sundquist WI, Frost A
  TITLE     Membrane constriction and thinning by sequential ESCRT-III 
            polymerization.
  JOURNAL   Nat Struct Mol Biol. 2020 Apr;27(4):392-399. doi: 
            10.1038/s41594-020-0404-x. Epub 2020 Apr 6.
  PUBMED    32251413
REFERENCE   2  (bases 1 to 5039)
  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"; doi: 
            "10.1038/s41594-020-0404-x"; journalName: "Nat Struct Mol Biol"; 
            date: "2020-04"; volume: "27"; issue: "4"; pages: "392-399"
COMMENT     Sequence Label: pGEX2T-TEV CHMP1B 186-199 L192A/L195A
FEATURES             Location/Qualifiers
     source          1..5039
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(180..185,186..201,202..208)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    216..232
                     /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             255..908
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     866..888
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             915..932
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             936..956
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(1086..1108)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1253..1271)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1339..1443
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1444..1512,1513..2304)
                     /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(1662..1681)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2475..3063
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2964..2983
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3217..3234
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3307..3384
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3385..4467
                     /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(3404..3423)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(4516..4521,4522..4539,4540..4546)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4554..4570
                     /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     4578..4594
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4578..4594
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(4590..5039,1..78)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(4610..4627)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4610..4626)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4619..4641)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 ttatcgactg cacggtgcac caatgcttct ggcgtcaggc agccatcgga agctgtggta
       61 tggctgtgca ggtcgtaaat cactgcataa ttcgtgtcgc tcaaggcgca ctcccgttct
      121 ggataatgtt ttttgcgccg acatcataac ggttctggca aatattctga aatgagctgt
      181 tgacaattaa tcatcggctc gtataatgtg tggaattgtg agcggataac aatttcacac
      241 aggaaacagt attcatgtcc cctatactag gttattggaa aattaagggc cttgtgcaac
      301 ccactcgact tcttttggaa tatcttgaag aaaaatatga agagcatttg tatgagcgcg
      361 atgaaggtga taaatggcga aacaaaaagt ttgaattggg tttggagttt cccaatcttc
      421 cttattatat tgatggtgat gttaaattaa cacagtctat ggccatcata cgttatatag
      481 ctgacaagca caacatgttg ggtggttgtc caaaagagcg tgcagagatt tcaatgcttg
      541 aaggagcggt tttggatatt agatacggtg tttcgagaat tgcatatagt aaagactttg
      601 aaactctcaa agttgatttt cttagcaagc tacctgaaat gctgaaaatg ttcgaagatc
      661 gtttatgtca taaaacatat ttaaatggtg atcatgtaac ccatcctgac ttcatgttgt
      721 atgacgctct tgatgttgtt ttatacatgg acccaatgtg cctggatgcg ttcccaaaat
      781 tagtttgttt taaaaaacgt attgaagcta tcccacaaat tgataagtac ttgaaatcca
      841 gcaagtatat agcatggcct ttgcagggct ggcaagccac gtttggtggt ggcgaccatc
      901 ctccaaaatc ggatctggtt ccgcgtggat ctccggaaaa cctgtatttc cagggccata
      961 tggatgaact gtctcagaga gcggcccgcg ctcgggatca agtgtgagga tcctcgagaa
     1021 ttctcgagaa ttcatcgtga ctgactgacg atctgcctcg cgcgtttcgg tgatgacggt
     1081 gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc
     1141 gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggcgcagcc
     1201 atgacccagt cacgtagcga tagcggagtg tataattctt gaagacgaaa gggcctcgtg
     1261 atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc
     1321 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     1381 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     1441 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     1501 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     1561 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     1621 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     1681 tcccgtgttg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     1741 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     1801 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     1861 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     1921 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     1981 atgcctgcag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     2041 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     2101 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
     2161 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     2221 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     2281 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     2341 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     2401 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     2461 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     2521 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     2581 aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag
     2641 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     2701 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     2761 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     2821 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     2881 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     2941 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     3001 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     3061 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     3121 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     3181 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
     3241 gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
     3301 aattccgaca ccatcgaatg gcgcaaaacc tttcgcggta tggcatgata gcgcccggaa
     3361 gagagtcaat tcagggtggt gaatgtgaaa ccagtaacgt tatacgatgt cgcagagtat
     3421 gccggtgtct cttatcagac cgtttcccgc gtggtgaacc aggccagcca cgtttctgcg
     3481 aaaacgcggg aaaaagtgga agcggcgatg gcggagctga attacattcc caaccgcgtg
     3541 gcacaacaac tggcgggcaa acagtcgttg ctgattggcg ttgccacctc cagtctggcc
     3601 ctgcacgcgc cgtcgcaaat tgtcgcggcg attaaatctc gcgccgatca actgggtgcc
     3661 agcgtggtgg tgtcgatggt agaacgaagc ggcgtcgaag cctgtaaagc ggcggtgcac
     3721 aatcttctcg cgcaacgcgt cagtgggctg atcattaact atccgctgga tgaccaggat
     3781 gccattgctg tggaagctgc ctgcactaat gttccggcgt tatttcttga tgtctctgac
     3841 cagacaccca tcaacagtat tattttctcc catgaagacg gtacgcgact gggcgtggag
     3901 catctggtcg cattgggtca ccagcaaatc gcgctgttag cgggcccatt aagttctgtc
     3961 tcggcgcgtc tgcgtctggc tggctggcat aaatatctca ctcgcaatca aattcagccg
     4021 atagcggaac gggaaggcga ctggagtgcc atgtccggtt ttcaacaaac catgcaaatg
     4081 ctgaatgagg gcatcgttcc cactgcgatg ctggttgcca acgatcagat ggcgctgggc
     4141 gcaatgcgcg ccattaccga gtccgggctg cgcgttggtg cggatatctc ggtagtggga
     4201 tacgacgata ccgaagacag ctcatgttat atcccgccgt taaccaccat caaacaggat
     4261 tttcgcctgc tggggcaaac cagcgtggac cgcttgctgc aactctctca gggccaggcg
     4321 gtgaagggca atcagctgtt gcccgtctca ctggtgaaaa gaaaaaccac cctggcgccc
     4381 aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag
     4441 gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtaagtt agctcactca
     4501 ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag
     4561 cggataacaa tttcacacag gaaacagcta tgaccatgat tacggattca ctggccgtcg
     4621 ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac
     4681 atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac
     4741 agttgcgcag cctgaatggc gaatggcgct ttgcctggtt tccggcacca gaagcggtgc
     4801 cggaaagctg gctggagtgc gatcttcctg aggccgatac tgtcgtcgtc ccctcaaact
     4861 ggcagatgca cggttacgat gcgcccatct acaccaacgt gacctatccc attacggtca
     4921 atccgccgtt tgttcccacg gagaatccga cgggttgtta ctcgctcaca tttaatgttg
     4981 atgaaagctg gctacaggaa ggccagacgc gaattatttt tgatggcgtt ggaattagc
//
