LOCUS       sequence-463003-        6059 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of a CHMP2B membrane binding mutant (L4D F5D) with the 
            fourth alpha helix deleted with a 3xHA tag..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6059)
  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 6059)
  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-d118-138-3xHA (Delta-alpha-4)
FEATURES             Location/Qualifiers
     source          1..6059
                     /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             1501..1527
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1531..1557
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1561..1587
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     promoter        complement(1600..1618)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1601..1618)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1638..1655)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1644..1868
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1914..2342
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2001..2020)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2211..2232
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2351..2371)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2356..2685
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2536..2671
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2598..2617
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2752..3546
                     /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(2806..2825)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3416..3435
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3720..3841
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3757..3776)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3811..3830
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3890..3906)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3890..3906)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3903..3925)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3914..3930
                     /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(3938..3944,3945..3962,3963..3968))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3983..4004
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4121..4138)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4292..4880)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4372..4391)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5051..5842,5843..5911))
                     /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     5674..5693
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5912..6016)
                     /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 aagatggatg gaggttcagg ttcaggtgga
     1261 ggatccgaag aaatgatcaa tgatacactt gatgacatct ttgacggttc tgatgacgaa
     1321 gaagaaagcc aggatattgt gaatcaagtt cttgatgaaa ttggaattga aatttctgga
     1381 aagatggcca aagctccatc agctgctcga agcttaccat ctgcctctac ttcaaaggct
     1441 acaatctcag atgaagagat tgaacggcaa ctcaaggctt taggagtaga tggaggatcc
     1501 tacccatacg acgtcccaga ctacgctgga tacccgtatg atgttccgga ttacgctggc
     1561 tacccatatg acgttccgga ttacgcttga gggccctatt ctatagtgtc acctaaatgc
     1621 tagagctcgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc
     1681 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa
     1741 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg
     1801 gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg
     1861 ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc
     1921 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     1981 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     2041 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     2101 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     2161 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     2221 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     2281 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     2341 ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc
     2401 agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc
     2461 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg
     2521 cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat
     2581 ggctgactaa ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc
     2641 cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct
     2701 tgtatatcca ttttcggatc tgatcaagag acaggatgag gatcgtttcg catgattgaa
     2761 caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac
     2821 tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg
     2881 cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaggacgag
     2941 gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt
     3001 gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg
     3061 tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg
     3121 catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga
     3181 gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag
     3241 gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat
     3301 ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt
     3361 tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg
     3421 gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt
     3481 tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc
     3541 ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac
     3601 gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg
     3661 acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca
     3721 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
     3781 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
     3841 atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt
     3901 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     3961 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     4021 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     4081 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     4141 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     4201 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     4261 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     4321 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     4381 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     4441 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     4501 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     4561 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     4621 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     4681 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     4741 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     4801 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     4861 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     4921 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     4981 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     5041 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     5101 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     5161 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     5221 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     5281 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     5341 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     5401 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     5461 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     5521 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     5581 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     5641 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     5701 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     5761 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     5821 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     5881 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     5941 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     6001 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac ggatcggga
//
