LOCUS       sequence-464217-        6092 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of a CHMP2B membrane binding mutant (L4D F5D) with the 
            loop connecting the second and third alpha helices deleted with a 
            3xHA tag..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6092)
  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 6092)
  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-d97-106-3xHA (Delta-alpha-2/3 
            loop)
FEATURES             Location/Qualifiers
     source          1..6092
                     /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             1534..1560
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1564..1590
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1594..1620
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     promoter        complement(1633..1651)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1634..1651)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1671..1688)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1677..1901
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1947..2375
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2034..2053)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2244..2265
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2384..2404)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2389..2718
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2569..2704
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2631..2650
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2785..3579
                     /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(2839..2858)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3449..3468
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3753..3874
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3790..3809)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3844..3863
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3923..3939)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3923..3939)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3936..3958)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3947..3963
                     /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(3971..3977,3978..3995,3996..4001))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4016..4037
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4154..4171)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4325..4913)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4405..4424)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5084..5875,5876..5944))
                     /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     5707..5726
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5945..6049)
                     /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 caaatgggag gttcaggttc aggtggagga
     1201 tccaaaacaa tgcaggcagt taacaagaag atggatccac aaaagacatt acaaacaatg
     1261 cagaatttcc agaaggaaaa catgaaaatg gaaatgactg aagaaatgat caatgataca
     1321 cttgatgaca tctttgacgg ttctgatgac gaagaggaaa gccaggatat tgtgaatcaa
     1381 gttcttgatg aaattggaat tgaaatttct ggaaagatgg ccaaagctcc atcagctgct
     1441 cgaagcttac catctgcctc tacttcaaag gctacaatct cagatgaaga gattgaacgg
     1501 caactcaagg ctttaggagt agatggagga tcctacccat acgacgtccc agactacgct
     1561 ggatacccgt atgatgttcc ggattacgct ggctacccat atgacgttcc ggattacgct
     1621 tgagggccct attctatagt gtcacctaaa tgctagagct cgctgatcag cctcgactgt
     1681 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga
     1741 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag
     1801 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga
     1861 agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac
     1921 cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg
     1981 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     2041 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     2101 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     2161 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     2221 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     2281 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     2341 aaatgagctg atttaacaaa aatttaacgc gaattaattc tgtggaatgt gtgtcagtta
     2401 gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     2461 tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     2521 atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta
     2581 actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca
     2641 gaggccgagg ccgcctctgc ctctgagcta ttccagaagt agtgaggagg cttttttgga
     2701 ggcctaggct tttgcaaaaa gctcccggga gcttgtatat ccattttcgg atctgatcaa
     2761 gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg
     2821 gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct
     2881 gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac
     2941 ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg
     3001 acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg
     3061 ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa
     3121 gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca
     3181 ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt
     3241 gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc
     3301 aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc
     3361 ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg
     3421 ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt
     3481 ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag
     3541 cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa
     3601 tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct
     3661 atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg
     3721 gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt
     3781 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     3841 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta
     3901 gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
     3961 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     4021 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     4081 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     4141 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     4201 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     4261 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     4321 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     4381 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     4441 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     4501 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     4561 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     4621 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     4681 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     4741 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     4801 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     4861 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     4921 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     4981 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     5041 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     5101 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     5161 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     5221 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     5281 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     5341 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     5401 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     5461 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     5521 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     5581 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     5641 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     5701 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     5761 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     5821 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     5881 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     5941 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     6001 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     6061 gaaaagtgcc acctgacgtc gacggatcgg ga
//
