LOCUS       sequence-245274-        5582 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses residues 4-199 with L to A mutations at residues 188 and 
            192 of CHMP1B with an N-terminal GST tag and a TEV cleavage site; 
            Internal ID: WISP 09-284.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5582)
  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 5582)
  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 4-199 L188A/L192A
FEATURES             Location/Qualifiers
     source          1..5582
                     /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(1629..1651)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1796..1814)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1882..1986
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1987..2055,2056..2847)
                     /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(2205..2224)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3018..3606
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3507..3526
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3760..3777
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3850..3927
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3928..5010
                     /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(3947..3966)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(5059..5064,5065..5082,5083..5089)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5097..5113
                     /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     5121..5137
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5121..5137
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(5133..5582,1..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(5153..5170)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5153..5169)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5162..5184)
                     /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 tggagaaaca cctgttcaac ctgaagttcg cggccaaaga actgagtagg agtgccaaaa
     1021 aatgcgataa ggaggaaaag gccgaaaagg ccaaaattga aaaggccatt cagaagggca
     1081 acatggaagt tgcgaggata cacgccgaaa atgccatccg ccagaagaac caggcggtga
     1141 atttcttgag aatgagtgcg cgagtcgatg cagtggctgc cagggtccag acggcggtga
     1201 cgatgggcaa ggtgaccaag tcgatggctg gtgtggttaa gtcgatggat gcgacattga
     1261 agaccatgaa tctggagaag atttctgctt tgatggacaa attcgagcac cagtttgaga
     1321 ctctggacgt ccagacgcag caaatggaag acacgatgag cagcacgacg acgctcacca
     1381 ctccccagaa ccaagtggat atgctgctcc aggaaatggc agatgaggcg ggcctcgacc
     1441 tcaacatgga gctgccgcag ggccagaccg gctccgtggg cacgagcgtg gcttcggcgg
     1501 agcaggatga agcgtctcag agagcggccc gccttcggga tcaagtgtaa ggatcctcga
     1561 gaattctcga gaattcatcg tgactgactg acgatctgcc tcgcgcgttt cggtgatgac
     1621 ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat
     1681 gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggcgca
     1741 gccatgaccc agtcacgtag cgatagcgga gtgtataatt cttgaagacg aaagggcctc
     1801 gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt
     1861 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     1921 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     1981 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     2041 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     2101 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     2161 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     2221 ttatcccgtg ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     2281 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     2341 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     2401 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     2461 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     2521 acgatgcctg cagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
     2581 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     2641 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
     2701 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
     2761 atctacacga cagggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     2821 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     2881 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     2941 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     3001 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     3061 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     3121 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg
     3181 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     3241 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     3301 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     3361 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     3421 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     3481 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     3541 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     3601 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     3661 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     3721 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     3781 gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     3841 ataaattccg acaccatcga atggcgcaaa acctttcgcg gtatggcatg atagcgcccg
     3901 gaagagagtc aattcagggt ggtgaatgtg aaaccagtaa cgttatacga tgtcgcagag
     3961 tatgccggtg tctcttatca gaccgtttcc cgcgtggtga accaggccag ccacgtttct
     4021 gcgaaaacgc gggaaaaagt ggaagcggcg atggcggagc tgaattacat tcccaaccgc
     4081 gtggcacaac aactggcggg caaacagtcg ttgctgattg gcgttgccac ctccagtctg
     4141 gccctgcacg cgccgtcgca aattgtcgcg gcgattaaat ctcgcgccga tcaactgggt
     4201 gccagcgtgg tggtgtcgat ggtagaacga agcggcgtcg aagcctgtaa agcggcggtg
     4261 cacaatcttc tcgcgcaacg cgtcagtggg ctgatcatta actatccgct ggatgaccag
     4321 gatgccattg ctgtggaagc tgcctgcact aatgttccgg cgttatttct tgatgtctct
     4381 gaccagacac ccatcaacag tattattttc tcccatgaag acggtacgcg actgggcgtg
     4441 gagcatctgg tcgcattggg tcaccagcaa atcgcgctgt tagcgggccc attaagttct
     4501 gtctcggcgc gtctgcgtct ggctggctgg cataaatatc tcactcgcaa tcaaattcag
     4561 ccgatagcgg aacgggaagg cgactggagt gccatgtccg gttttcaaca aaccatgcaa
     4621 atgctgaatg agggcatcgt tcccactgcg atgctggttg ccaacgatca gatggcgctg
     4681 ggcgcaatgc gcgccattac cgagtccggg ctgcgcgttg gtgcggatat ctcggtagtg
     4741 ggatacgacg ataccgaaga cagctcatgt tatatcccgc cgttaaccac catcaaacag
     4801 gattttcgcc tgctggggca aaccagcgtg gaccgcttgc tgcaactctc tcagggccag
     4861 gcggtgaagg gcaatcagct gttgcccgtc tcactggtga aaagaaaaac caccctggcg
     4921 cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga
     4981 caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtaa gttagctcac
     5041 tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt
     5101 gagcggataa caatttcaca caggaaacag ctatgaccat gattacggat tcactggccg
     5161 tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag
     5221 cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc
     5281 aacagttgcg cagcctgaat ggcgaatggc gctttgcctg gtttccggca ccagaagcgg
     5341 tgccggaaag ctggctggag tgcgatcttc ctgaggccga tactgtcgtc gtcccctcaa
     5401 actggcagat gcacggttac gatgcgccca tctacaccaa cgtgacctat cccattacgg
     5461 tcaatccgcc gtttgttccc acggagaatc cgacgggttg ttactcgctc acatttaatg
     5521 ttgatgaaag ctggctacag gaaggccaga cgcgaattat ttttgatggc gttggaatta
     5581 gc
//
