LOCUS       sequence-248801-        6192 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses residues 1-199 of CHMP1B from a His-SUMO bacterial 
            expression vector. Internal ID: WISP 18-24.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6192)
  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 6192)
  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: pCA528 CHMP1B 1-199
FEATURES             Location/Qualifiers
     source          1..6192
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             771..788
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(841..859)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      855..902
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      939..1394
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1026..1045)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1236..1257
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1486..2301)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(1486..2301)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2209..2228
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2293..2312
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2423..3011
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2912..2931
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3165..3182
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3197..3336
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3262..3281)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3422..3444
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3438..3629)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4438..5520)
                     /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
                     MSGFQQTMKMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     5482..5501
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5521..5598)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5804..5823
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5907..5926
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5907..5925
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5926..5950
                     /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)."
     RBS             5965..5987
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             6001..6018
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             join(6022..6192,1..120)
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="SDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLR
                     RLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
ORIGIN
        1 gaaatggact ccttaagatt cttgtacgac ggtattagaa ttcaagctga tcagacccct
       61 gaagatttgg acatggagga taacgatatt attgaggctc acagagaaca gattggtggg
      121 atgagcaaca tggagaaaca cctgttcaac ctgaagttcg cggccaaaga actgagtagg
      181 agtgccaaaa aatgcgataa ggaggaaaag gccgaaaagg ccaaaattga aaaggccatt
      241 cagaagggca acatggaagt tgcgaggata cacgccgaaa atgccatccg ccagaagaac
      301 caggcggtga atttcttgag aatgagtgcg cgagtcgatg cagtggctgc cagggtccag
      361 acggcggtga cgatgggcaa ggtgaccaag tcgatggctg gtgtggttaa gtcgatggat
      421 gcgacattga agaccatgaa tctggagaag atttctgctt tgatggacaa attcgagcac
      481 cagtttgaga ctctggacgt ccagacgcag caaatggaag acacgatgag cagcacgacg
      541 acgctcacca ctccccagaa ccaagtggat atgctgctcc aggaaatggc agatgaggcg
      601 ggcctcgacc tcaacatgga gctgccgcag ggccagaccg gctccgtggg cacgagcgtg
      661 gcttcggcgg agcaggatga actgtctcag agactggccc gccttcggga tcaagtgtaa
      721 gaccggatcc gaattcgagc tccgtcgaca agcttgcggc cgcactcgag caccaccacc
      781 accaccactg agatccggct gctaacaaag cccgaaagga agctgagttg gctgctgcca
      841 ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt
      901 tgctgaaagg aggaactata tccggattgg cgaatgggac gcgccctgta gcggcgcatt
      961 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     1021 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     1081 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     1141 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     1201 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     1261 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     1321 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
     1381 aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     1441 tttttctaaa tacattcaaa tatgtatccg ctcatgaatt aattcttaga aaaactcatc
     1501 gagcatcaaa tgaaactgca atttattcat atcaggatta tcaataccat atttttgaaa
     1561 aagccgtttc tgtaatgaag gagaaaactc accgaggcag ttccatagga tggcaagatc
     1621 ctggtatcgg tctgcgattc cgactcgtcc aacatcaata caacctatta atttcccctc
     1681 gtcaaaaata aggttatcaa gtgagaaatc accatgagtg acgactgaat ccggtgagaa
     1741 tggcaaaagt ttatgcattt ctttccagac ttgttcaaca ggccagccat tacgctcgtc
     1801 atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct gagcgagacg
     1861 aaatacgcga tcgctgttaa aaggacaatt acaaacagga atcgaatgca accggcgcag
     1921 gaacactgcc agcgcatcaa caatattttc acctgaatca ggatattctt ctaatacctg
     1981 gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat gcatcatcag gagtacggat
     2041 aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc cagtttagtc tgaccatctc
     2101 atctgtaaca tcattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatc
     2161 gggcttccca tacaatcgat agattgtcgc acctgattgc ccgacattat cgcgagccca
     2221 tttataccca tataaatcag catccatgtt ggaatttaat cgcggcctag agcaagacgt
     2281 ttcccgttga atatggctca taacacccct tgtattactg tttatgtaag cagacagttt
     2341 tattgttcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     2401 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     2461 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     2521 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt
     2581 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     2641 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     2701 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     2761 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     2821 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     2881 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     2941 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     3001 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     3061 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     3121 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     3181 aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     3241 accgcaatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac
     3301 actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct
     3361 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     3421 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg
     3481 cggtaaagct catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg
     3541 tccagctcgt tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg
     3601 ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc
     3661 atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat
     3721 gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg
     3781 gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt
     3841 ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct
     3901 gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct
     3961 caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca
     4021 ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg
     4081 atcatgcgca cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt
     4141 ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca
     4201 agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag
     4261 agcgctgccg gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg
     4321 acgatagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc
     4381 atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca
     4441 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     4501 gcgtggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac
     4561 gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac
     4621 gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca
     4681 tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc
     4741 ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc
     4801 agtgggaacg atgccctcat tcagcatttt catggtttgt tgaaaaccgg acatggcact
     4861 ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca
     4921 gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg
     4981 ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa
     5041 aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt
     5101 gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc
     5161 actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg
     5221 ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc
     5281 gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga
     5341 ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc
     5401 cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga
     5461 aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac
     5521 attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt
     5581 gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga
     5641 agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca
     5701 aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac
     5761 aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat
     5821 aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga
     5881 ggatcgagat ctcgatcccg cgaaattaat acgactcact ataggggaat tgtgagcgga
     5941 taacaattcc cctctagaaa taattttgtt taactttaag aaggagatat acatattggt
     6001 catcaccatc atcaccatgg ttcggactca gaagtcaatc aagaagctaa gccagaggtc
     6061 aagccagaag tcaagcctga gactcacatc aatttaaagg tgtccgatgg atcttcagag
     6121 atcttcttca agatcaaaaa gaccactcct ttaagaaggc tgatggaagc gttcgctaaa
     6181 agacagggta ag
//
