LOCUS       sequence-470414-        5870 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of a CHMP2B membrane binding mutant (L4D F5D) with the 
            second through fourth helices deleted with a 3xHA tag..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5870)
  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 5870)
  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-d55-138-3xHA (Delta-alpha-2-4)
FEATURES             Location/Qualifiers
     source          1..5870
                     /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             1312..1338
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1342..1368
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1372..1398
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     promoter        complement(1411..1429)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1412..1429)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1449..1466)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1455..1679
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1725..2153
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1812..1831)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2022..2043
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2162..2182)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2167..2496
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2347..2482
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2409..2428
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2563..3357
                     /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(2617..2636)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3227..3246
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3531..3652
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3568..3587)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3622..3641
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3701..3717)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3701..3717)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3714..3736)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3725..3741
                     /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(3749..3755,3756..3773,3774..3779))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3794..3815
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3932..3949)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4103..4691)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4183..4202)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4862..5653,5654..5722))
                     /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     5485..5504
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5723..5827)
                     /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 ggaggttcag gttcaggtgg aggatccgaa
     1081 gaaatgatca atgatacact tgatgacatc tttgacggtt ctgatgacga agaagaaagc
     1141 caggatattg tgaatcaagt tcttgatgaa attggaattg aaatttctgg aaagatggcc
     1201 aaagctccat cagctgctcg aagcttacca tctgcctcta cttcaaaggc tacaatctca
     1261 gatgaagaga ttgaacggca actcaaggct ttaggagtag atggaggatc ctacccatac
     1321 gacgtcccag actacgctgg atacccgtat gatgttccgg attacgctgg ctacccatat
     1381 gacgttccgg attacgcttg agggccctat tctatagtgt cacctaaatg ctagagctcg
     1441 ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt
     1501 gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat
     1561 tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag
     1621 caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc
     1681 ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc cctgtagcgg
     1741 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     1801 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     1861 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     1921 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     1981 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     2041 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     2101 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attaattctg
     2161 tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     2221 caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca
     2281 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact
     2341 ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta
     2401 atttttttta tttatgcaga ggccgaggcc gcctctgcct ctgagctatt ccagaagtag
     2461 tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc
     2521 attttcggat ctgatcaaga gacaggatga ggatcgtttc gcatgattga acaagatgga
     2581 ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa
     2641 cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt
     2701 ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg
     2761 ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa
     2821 gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac
     2881 cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt
     2941 gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact
     3001 cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg
     3061 ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg
     3121 acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc
     3181 atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt
     3241 gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc
     3301 gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgagcg
     3361 ggactctggg gttcgaaatg accgaccaag cgacgcccaa cctgccatca cgagatttcg
     3421 attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct
     3481 ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta
     3541 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
     3601 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct
     3661 gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt
     3721 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
     3781 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
     3841 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     3901 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     3961 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     4021 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     4081 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     4141 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     4201 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     4261 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     4321 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     4381 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     4441 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     4501 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     4561 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     4621 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     4681 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     4741 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     4801 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     4861 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     4921 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     4981 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     5041 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     5101 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     5161 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     5221 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     5281 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     5341 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     5401 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     5461 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     5521 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     5581 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     5641 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     5701 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     5761 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     5821 ttccgcgcac atttccccga aaagtgccac ctgacgtcga cggatcggga
//
