LOCUS       sequence-463795-        6098 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of a CHMP2B membrane binding mutant (L4D F5D) with the 
            third alpha helix deleted with a 3xHA tag..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6098)
  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 6098)
  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-d106-113-3xHA (Delta-alpha-3)
FEATURES             Location/Qualifiers
     source          1..6098
                     /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             1540..1566
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1570..1596
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1600..1626
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     promoter        complement(1639..1657)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1640..1657)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1677..1694)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1683..1907
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1953..2381
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2040..2059)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2250..2271
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2390..2410)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2395..2724
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2575..2710
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2637..2656
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2791..3585
                     /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(2845..2864)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3455..3474
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3759..3880
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3796..3815)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3850..3869
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3929..3945)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3929..3945)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3942..3964)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3953..3969
                     /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(3977..3983,3984..4001,4002..4007))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4022..4043
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4160..4177)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4331..4919)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4411..4430)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5090..5881,5882..5950))
                     /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     5713..5732
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5951..6055)
                     /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 acaggaggtt caggttcagg tggaggatcc aagaagatgg atccacaaaa gacattacaa
     1261 acaatgcaga atttccagaa ggaaaacatg aaaatggaaa tgactgaaga aatgatcaat
     1321 gatacacttg atgacatctt tgacggttct gatgacgaag aagaaagcca ggatattgtg
     1381 aatcaagttc ttgatgaaat tggaattgaa atttctggaa agatggccaa agctccatca
     1441 gctgctcgaa gcttaccatc tgcctctact tcaaaggcta caatctcaga tgaagagatt
     1501 gaacggcaac tcaaggcttt aggagtagat ggaggatcct acccatacga cgtcccagac
     1561 tacgctggat acccgtatga tgttccggat tacgctggct acccatatga cgttccggat
     1621 tacgcttgag ggccctattc tatagtgtca cctaaatgct agagctcgct gatcagcctc
     1681 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
     1741 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg
     1801 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga
     1861 ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga
     1921 aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc
     1981 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     2041 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     2101 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     2161 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     2221 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     2281 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     2341 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt
     2401 cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     2461 ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     2521 caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg
     2581 cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt
     2641 tatgcagagg ccgaggccgc ctctgcctct gagctattcc agaagtagtg aggaggcttt
     2701 tttggaggcc taggcttttg caaaaagctc ccgggagctt gtatatccat tttcggatct
     2761 gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt
     2821 tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc
     2881 tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag
     2941 accgacctgt ccggtgccct gaatgaactg caggacgagg cagcgcggct atcgtggctg
     3001 gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac
     3061 tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc
     3121 gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc
     3181 tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc
     3241 ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg
     3301 ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat
     3361 gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc
     3421 cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa
     3481 gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat
     3541 tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt
     3601 tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat tccaccgccg
     3661 ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc
     3721 agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata
     3781 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     3841 attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga
     3901 cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     3961 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     4021 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     4081 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     4141 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     4201 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     4261 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     4321 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     4381 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     4441 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     4501 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     4561 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     4621 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     4681 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     4741 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     4801 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     4861 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     4921 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     4981 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     5041 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     5101 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     5161 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     5221 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     5281 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     5341 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     5401 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     5461 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     5521 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     5581 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     5641 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     5701 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     5761 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     5821 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     5881 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     5941 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     6001 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     6061 ttccccgaaa agtgccacct gacgtcgacg gatcggga
//
