LOCUS       sequence-469261-        5996 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of a CHMP2B membrane binding mutant (L4D F5D) with the 
            VPS4-binding region deleted with a 3xHA tag..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5996)
  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 5996)
  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-d201-213-HA (Delta-VPS4 binding)
FEATURES             Location/Qualifiers
     source          1..5996
                     /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             1498..1524
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     promoter        complement(1537..1555)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1538..1555)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1575..1592)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1581..1805
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1851..2279
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1938..1957)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2148..2169
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2288..2308)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2293..2622
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2473..2608
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2535..2554
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2689..3483
                     /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(2743..2762)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3353..3372
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3657..3778
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3694..3713)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3748..3767
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3827..3843)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3827..3843)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3840..3862)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3851..3867
                     /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(3875..3881,3882..3899,3900..3905))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3920..3941
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4058..4075)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4229..4817)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4309..4328)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4988..5779,5780..5848))
                     /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     5611..5630
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5849..5953)
                     /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 agttacttct
     1141 atgtctacac aaacaaaagt gatgaattcc 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 tctcagatgg aggcagctac
     1501 ccatacgacg tcccagacta cgcttgaggg ccctattcta tagtgtcacc taaatgctag
     1561 agctcgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc
     1621 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga
     1681 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca
     1741 ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc
     1801 tatggcttct gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg
     1861 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     1921 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     1981 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     2041 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg
     2101 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     2161 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt
     2221 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaatta
     2281 attctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga
     2341 agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc
     2401 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc
     2461 ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc
     2521 tgactaattt tttttattta tgcagaggcc gaggccgcct ctgcctctga gctattccag
     2581 aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctccc gggagcttgt
     2641 atatccattt tcggatctga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa
     2701 gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg
     2761 gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc
     2821 ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca ggacgaggca
     2881 gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc
     2941 actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca
     3001 tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat
     3061 acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca
     3121 cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg
     3181 ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc
     3241 gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct
     3301 ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct
     3361 acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac
     3421 ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc
     3481 tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag
     3541 atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg
     3601 ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccaact
     3661 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     3721 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
     3781 atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc
     3841 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     3901 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     3961 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     4021 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     4081 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     4141 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     4201 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     4261 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     4321 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     4381 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     4441 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     4501 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     4561 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     4621 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     4681 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     4741 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     4801 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     4861 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     4921 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     4981 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     5041 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     5101 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     5161 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     5221 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     5281 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     5341 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     5401 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     5461 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     5521 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     5581 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     5641 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     5701 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     5761 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     5821 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     5881 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     5941 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtcgacgga tcggga
//
