LOCUS       sequence-245268-        5414 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses residues 1-143 of CHMP1B with an N-terminal GST tag and a 
            TEV cleavage site; Internal ID: WISP 09-278.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5414)
  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 5414)
  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 1-143
FEATURES             Location/Qualifiers
     source          1..5414
                     /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(1461..1483)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1628..1646)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1714..1818
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1819..1887,1888..2679)
                     /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(2037..2056)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2850..3438
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3339..3358
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3592..3609
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3682..3759
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3760..4842
                     /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(3779..3798)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(4891..4896,4897..4914,4915..4921)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4929..4945
                     /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     4953..4969
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4953..4969
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(4965..5414,1..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(4985..5002)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4985..5001)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4994..5016)
                     /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 acgctcacct
     1381 gaggatcctc gagaattctc gagaattcat cgtgactgac tgacgatctg cctcgcgcgt
     1441 ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt
     1501 ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg
     1561 tgtcggggcg cagccatgac ccagtcacgt agcgatagcg gagtgtataa ttcttgaaga
     1621 cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct
     1681 tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     1741 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     1801 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     1861 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     1921 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     1981 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     2041 tgtggcgcgg tattatcccg tgttgacgcc gggcaagagc aactcggtcg ccgcatacac
     2101 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     2161 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     2221 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     2281 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     2341 gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact attaactggc
     2401 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     2461 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     2521 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     2581 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     2641 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     2701 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     2761 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     2821 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     2881 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     2941 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt
     3001 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     3061 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     3121 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     3181 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     3241 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     3301 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     3361 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     3421 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     3481 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     3541 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     3601 gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt
     3661 atttcacacc gcataaattc cgacaccatc gaatggcgca aaacctttcg cggtatggca
     3721 tgatagcgcc cggaagagag tcaattcagg gtggtgaatg tgaaaccagt aacgttatac
     3781 gatgtcgcag agtatgccgg tgtctcttat cagaccgttt cccgcgtggt gaaccaggcc
     3841 agccacgttt ctgcgaaaac gcgggaaaaa gtggaagcgg cgatggcgga gctgaattac
     3901 attcccaacc gcgtggcaca acaactggcg ggcaaacagt cgttgctgat tggcgttgcc
     3961 acctccagtc tggccctgca cgcgccgtcg caaattgtcg cggcgattaa atctcgcgcc
     4021 gatcaactgg gtgccagcgt ggtggtgtcg atggtagaac gaagcggcgt cgaagcctgt
     4081 aaagcggcgg tgcacaatct tctcgcgcaa cgcgtcagtg ggctgatcat taactatccg
     4141 ctggatgacc aggatgccat tgctgtggaa gctgcctgca ctaatgttcc ggcgttattt
     4201 cttgatgtct ctgaccagac acccatcaac agtattattt tctcccatga agacggtacg
     4261 cgactgggcg tggagcatct ggtcgcattg ggtcaccagc aaatcgcgct gttagcgggc
     4321 ccattaagtt ctgtctcggc gcgtctgcgt ctggctggct ggcataaata tctcactcgc
     4381 aatcaaattc agccgatagc ggaacgggaa ggcgactgga gtgccatgtc cggttttcaa
     4441 caaaccatgc aaatgctgaa tgagggcatc gttcccactg cgatgctggt tgccaacgat
     4501 cagatggcgc tgggcgcaat gcgcgccatt accgagtccg ggctgcgcgt tggtgcggat
     4561 atctcggtag tgggatacga cgataccgaa gacagctcat gttatatccc gccgttaacc
     4621 accatcaaac aggattttcg cctgctgggg caaaccagcg tggaccgctt gctgcaactc
     4681 tctcagggcc aggcggtgaa gggcaatcag ctgttgcccg tctcactggt gaaaagaaaa
     4741 accaccctgg cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg
     4801 cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt
     4861 aagttagctc actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt
     4921 gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgg
     4981 attcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta
     5041 atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg
     5101 atcgcccttc ccaacagttg cgcagcctga atggcgaatg gcgctttgcc tggtttccgg
     5161 caccagaagc ggtgccggaa agctggctgg agtgcgatct tcctgaggcc gatactgtcg
     5221 tcgtcccctc aaactggcag atgcacggtt acgatgcgcc catctacacc aacgtgacct
     5281 atcccattac ggtcaatccg ccgtttgttc ccacggagaa tccgacgggt tgttactcgc
     5341 tcacatttaa tgttgatgaa agctggctac aggaaggcca gacgcgaatt atttttgatg
     5401 gcgttggaat tagc
//
