LOCUS       sequence-365323-        6554 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of CHMP1B (S2C mutation).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6554)
  AUTHORS   Cada AK, Pavlin MR, Castillo JP, Tong AB, Larsen KP, Ren X, Yokom 
            AL, Tsai FC, Shiah JV, Bassereau PM, Bustamante CJ, Hurley JH
  TITLE     Friction-driven membrane scission by the human ESCRT-III proteins 
            CHMP1B and IST1.
  JOURNAL   Proc Natl Acad Sci U S A. 2022 Jul 19;119(29):e2204536119. doi: 
            10.1073/pnas.2204536119. Epub 2022 Jul 11.
  PUBMED    35858336
REFERENCE   2  (bases 1 to 6554)
  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: "Proc Natl 
            Acad Sci U S A."; date: "2022-07-19"; volume: "119(29):e2204536119. 
            doi"; pages: " 10.1073/pnas.2204536119. Epub 2022 Jul 11"
COMMENT     Sequence Label: His-MBP-CHMP1B, S2C
FEATURES             Location/Qualifiers
     source          1..6554
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     815..834
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     918..937
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        918..936
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             1025..1047
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1067..1084
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1094..2194
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     2168..2191
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             2249..2269
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(3001..3019)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3015..3062
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(3428..3433,3434..3450,3451..3456))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(3483..3501)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3569..3673
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3674..3742,3743..4534)
                     /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(3892..3911)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4705..5293
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5194..5213
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5447..5464
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5479..5618
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5544..5563)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5704..5726
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5720..5911)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 tcgaggtggc ccggctccat gcaccgcgac gcaacgcggg gaggcagaca aggtataggg
       61 cggcgcctac aatccatgcc aacccgttcc atgtgctcgc cgaggcggca taaatcgccg
      121 tgacgatcag cggtccaatg atcgaagtta ggctggtaag agccgcgagc gatccttgaa
      181 gctgtccctg atggtcgtca tctacctgcc tggacagcat ggcctgcaac gcgggcatcc
      241 cgatgccgcc ggaagcgaga agaatcataa tggggaaggc catccagcct cgcgtcgcga
      301 acgccagcaa gacgtagccc agcgcgtcgg ccgccatgcc ggcgataatg gcctgcttct
      361 cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg agcgagggcg tgcaagattc
      421 cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga
      481 aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg catgataaag aagacagtca
      541 taagtgcggc gacgatagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg
      601 ctctcaaggg catcggtcga cgctctccct tatgcgactc ctgcattagg aagcagccca
      661 gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg
      721 cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca
      781 tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag
      841 caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga
      901 tctcgatccc gcgaaattaa tacgactcac tatagggaga ccacaacggt ttccctctag
      961 tgccggctcc ggagagctct ttaattaagc ggccgccctg caggactcga gttctagaaa
     1021 taattttgtt taactttaag aaggagatat acatatgaaa tcttctcacc atcaccatca
     1081 ccatggttct tctatgaaaa tcgaagaagg taaactggta atctggatta acggcgataa
     1141 aggctataac ggtctcgctg aagtcggtaa gaaattcgag aaagataccg gaattaaagt
     1201 caccgttgag catccggata aactggaaga gaaattccca caggttgcgg caactggcga
     1261 tggccctgac attatcttct gggcacacga ccgctttggt ggctacgctc aatctggcct
     1321 gttggctgaa atcaccccgg acaaagcgtt ccaggacaag ctgtatccgt ttacctggga
     1381 tgccgtacgt tacaacggca agctgattgc ttacccgatc gctgttgaag cgttatcgct
     1441 gatttataac aaagatctgc tgccgaaccc gccaaaaacc tgggaagaga tcccggcgct
     1501 ggataaagaa ctgaaagcga aaggtaagag cgcgctgatg ttcaacctgc aagaaccgta
     1561 cttcacctgg ccgctgattg ctgctgacgg gggttatgcg ttcaagtatg aaaacggcaa
     1621 gtacgacatt aaagacgtgg gcgtggataa cgctggcgcg aaagcgggtc tgaccttcct
     1681 ggttgacctg attaaaaaca aacacatgaa tgcagacacc gattactcca tcgcagaagc
     1741 tgcctttaat aaaggcgaaa cagcgatgac catcaacggc ccgtgggcat ggtccaacat
     1801 cgacaccagc aaagtgaatt atggtgtaac ggtactgccg accttcaagg gtcaaccatc
     1861 caaaccgttc gttggcgtgc tgagcgcagg tattaacgcc gccagtccga acaaagagct
     1921 ggcaaaagag ttcctcgaaa actatctgct gactgatgaa ggtctggaag cggttaataa
     1981 agacaaaccg ctgggtgccg tagcgctgaa gtcttacgag gaagagttgg cgaaagatcc
     2041 acgtattgcc gccactatgg aaaacgccca gaaaggtgaa atcatgccga acatcccgca
     2101 gatgtccgct ttctggtatg ccgtgcgtac tgcggtgatc aacgccgcca gcggtcgtca
     2161 gactgtcgat gaagccctga aagacgcgca gactaattcg agctcgaaca acaacaacaa
     2221 taacaataac aacaacctcg ggatcgagga aaacctgtac ttccaatcca atgcaatgtg
     2281 caacatggag aaacacctgt tcaacctgaa gttcgcggcc aaagaactga gtaggagtgc
     2341 caaaaaatgc gataaggagg aaaaggccga aaaggccaaa attaaaaagg ccattcagaa
     2401 gggcaacatg gaagttgcga ggatacacgc cgaaaatgcc atccgccaga agaaccaggc
     2461 ggtgaatttc ttgagaatga gtgcgcgagt cgatgcagtg gctgccaggg tccagacggc
     2521 ggtgacgatg ggcaaggtga ccaagtcgat ggctggtgtg gttaagtcga tggatgcgac
     2581 attgaagacc atgaatctgg agaagatttc tgctttgatg gacaaattcg agcaccagtt
     2641 tgagactctg gacgtccaga cgcagcaaat ggaagacacg atgagcagca cgacgacgct
     2701 caccactccc cagaaccaag tggatatgct gctccaggaa atggcagatg aggcgggcct
     2761 cgacctcaac atggagctgc cgcagggcca gaccggctcc gtgggcacga gcgtggcttc
     2821 ggcggagcag gatgaactgt ctcagagact ggcccgcctt cgggatcaag tgtaataaca
     2881 ttggaagtgg ataacggatc cgcgatcgcg gcgcgccacc tggtggccgg ccggtaccac
     2941 gcgtgcgcgc tgatccggct gctaacaaag cccgaaagga agctgagttg gctgctgcca
     3001 ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt
     3061 tgctgaaagg aggaactata tccggatatc cacaggacgg gtgtggtcgc catgatcgcg
     3121 tagtcgatag tggctccaag tagcgaagcg agcaggactg ggcggcggcc aaagcggtcg
     3181 gacagtgctc cgagaacggg tgcgcataga aattgcatca acgcatatag cgctagcagc
     3241 acgccatagt gactggcgat gctgtcggaa tggacgatat cccgcaagag gcccggcagt
     3301 accggcataa ccaagcctat gcctacagca tccagggtga cggtgccgag gatgacgatg
     3361 agcgcattgt tagatttcat acacggtgcc tgactgcgtt agcaatttaa ctgtgataaa
     3421 ctaccgcatt aaagcttatc gatgataagc tgtcaaacat gagaattctt gaagacgaaa
     3481 gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac
     3541 gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     3601 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataacattg
     3661 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
     3721 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
     3781 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga
     3841 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg
     3901 cgcggtatta tcccgtgttg acgccgggca agagcaactc ggtcgccgca tacactattc
     3961 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
     4021 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
     4081 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
     4141 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
     4201 tgacaccacg atgcctgcag caatggcaac aacgttgcgc aaactattaa ctggcgaact
     4261 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
     4321 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
     4381 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
     4441 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     4501 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
     4561 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     4621 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     4681 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     4741 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     4801 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt
     4861 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     4921 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     4981 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     5041 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     5101 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     5161 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     5221 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     5281 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     5341 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     5401 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     5461 cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     5521 acaccgcaat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat
     5581 acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg
     5641 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     5701 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc
     5761 tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg
     5821 cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata aagcgggcca
     5881 tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt
     5941 tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg gttactgatg
     6001 atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta tggatgcggc
     6061 gggaccagag aaaaatcact cagggtcaat gccagcgctt cgttaataca gatgtaggtg
     6121 ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg gtgcagggcg
     6181 ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagaccatt catgttgttg
     6241 ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt
     6301 cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac gacaggagca
     6361 cgatcatgcg cacccgtggc caggacccaa cgctgcccga gatgcgccgc gtgcggctgc
     6421 tggagatggc ggacgcgatg gatatgttct gccaagggtt ggtttgcgca ttcacagttc
     6481 tccgcaagaa ttgattggct ccaattcttg gagtggtgaa tccgttagcg aggtgccgcc
     6541 ggcttccatt cagg
//
