LOCUS       sequence-486101-        6095 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of a CHMP2B membrane binding mutant (L4D F5D) with V82E, 
            M85E, and M92E mutations and a 3xHA tag..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6095)
  AUTHORS   Sitron CS, Trinkaus VA, Galesic A, Garhammer M, Yuste-Checa P, 
            Dransfeld U, Feigenbutz D, Zhang J, Ivashko L, Dudanova I, Harper 
            JW, Hartl FU
  TITLE     alpha-Synuclein aggregates inhibit ESCRT-III through sequestration 
            and collateral degradation.
  JOURNAL   Mol Cell. 2025 Sep 18;85(18):3505-3523.e17. doi: 
            10.1016/j.molcel.2025.08.022. Epub 2025 Sep 10.
  PUBMED    40934925
REFERENCE   2  (bases 1 to 6095)
  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: 
            "10.1016/j.molcel.2025.08.022"; journalName: "Mol Cell"; date: 
            "2025-09-18- 18"; volume: "85"; issue: "18"; pages: "3505-3523.e17"
COMMENT     Sequence Label: pCMV CHMP2B-L4D,F5D-V82E M85E M92E-3XHA
FEATURES             Location/Qualifiers
     source          1..6095
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(32..51)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        223..602
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        603..806
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     757..777
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     851..870
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        851..869
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1537..1563
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1567..1593
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1597..1623
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     promoter        complement(1636..1654)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1637..1654)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1674..1691)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1680..1904
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1950..2378
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2037..2056)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2247..2268
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2387..2407)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2392..2721
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2572..2707
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2634..2653
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2788..3582
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2842..2861)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3452..3471
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3756..3877
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3793..3812)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3847..3866
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3926..3942)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3926..3942)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3939..3961)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3950..3966
                     /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)."
     promoter        complement(join(3974..3980,3981..3998,3999..4004))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4019..4040
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4157..4174)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4328..4916)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4408..4427)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5087..5878,5879..5947))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5710..5729
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5948..6052)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gatctcccga tcccctatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
       61 gccagtatct gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt
      121 aagctacaac aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt agggttaggc
      181 gttttgcgct gcttcgcgat gtacgggcca gatatacgcg ttgacattga ttattgacta
      241 gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg
      301 ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga
      361 cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat
      421 gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa
      481 gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca
      541 tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc gctattacca
      601 tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat
      661 ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg
      721 actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac
      781 ggtgggaggt ctatataagc agagctctct ggctaactag agaacccact gcttactggc
      841 ttatcgaaat taatacgact cactataggg agacccaagc tggagaccat ggcgtccgac
      901 gacaagaaga aaaccgtgga tgatgtaata aaggaacaga atcgagagtt acgaggtaca
      961 cagagggcta taatcagaga tcgagcagct ttagagaaac aagaaaaaca gctggaatta
     1021 gaaattaaga aaatggccaa gattggtaat aaggaagctt gcaaagtttt agccaaacaa
     1081 cttgtgcatc tacggaaaca gaagacgaga acttttgctg taagttcaaa agaaacttct
     1141 gagtctacac aaacaaaagt ggagaattcc caaatgaaga tggctggagc aatgtctacc
     1201 acagcaaaaa caatgcaggc agttaacaag aagatggatc cacaaaagac attacaaaca
     1261 atgcagaatt tccagaagga aaacatgaaa atggaaatga ctgaagaaat gatcaatgat
     1321 acacttgatg acatctttga cggttctgat gacgaagaag aaagccagga tattgtgaat
     1381 caagttcttg atgaaattgg aattgaaatt tctggaaaga tggccaaagc tccatcagct
     1441 gctcgaagct taccatctgc ctctacttca aaggctacaa tctcagatga agagattgaa
     1501 cggcaactca aggctttagg agtagatgga ggatcctacc catacgacgt cccagactac
     1561 gctggatacc cgtatgatgt tccggattac gctggctacc catatgacgt tccggattac
     1621 gcttgagggc cctattctat agtgtcacct aaatgctaga gctcgctgat cagcctcgac
     1681 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
     1741 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
     1801 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
     1861 ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag
     1921 aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc
     1981 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     2041 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     2101 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     2161 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     2221 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     2281 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     2341 aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag
     2401 ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     2461 aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     2521 agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc
     2581 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
     2641 gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt
     2701 ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt cggatctgat
     2761 caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct
     2821 ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc
     2881 tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc
     2941 gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc
     3001 acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg
     3061 ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag
     3121 aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc
     3181 ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt
     3241 cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc
     3301 gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc
     3361 tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg
     3421 ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag
     3481 cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg
     3541 cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg
     3601 aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct
     3661 tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc
     3721 gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg
     3781 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     3841 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct
     3901 ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     3961 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     4021 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     4081 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     4141 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     4201 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     4261 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     4321 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     4381 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     4441 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     4501 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     4561 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     4621 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     4681 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     4741 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     4801 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     4861 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     4921 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     4981 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     5041 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     5101 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     5161 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     5221 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     5281 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     5341 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     5401 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     5461 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     5521 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     5581 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     5641 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     5701 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     5761 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     5821 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     5881 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     5941 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     6001 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     6061 cccgaaaagt gccacctgac gtcgacggat cggga
//
