LOCUS       sequence-477196-        5993 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of a CHMP2B membrane binding mutant (L4D F5D) with the 
            first alpha helix deleted with a 3xHA tag..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5993)
  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 5993)
  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-d10-52-3xHA (Delta-alpha-1)
FEATURES             Location/Qualifiers
     source          1..5993
                     /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             1435..1461
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1465..1491
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1495..1521
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     promoter        complement(1534..1552)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1535..1552)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1572..1589)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1578..1802
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1848..2276
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1935..1954)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2145..2166
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2285..2305)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2290..2619
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2470..2605
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2532..2551
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2686..3480
                     /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(2740..2759)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3350..3369
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3654..3775
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3691..3710)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3745..3764
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3824..3840)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3824..3840)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3837..3859)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3848..3864
                     /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(3872..3878,3879..3896,3897..3902))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3917..3938
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4055..4072)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4226..4814)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4306..4325)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4985..5776,5777..5845))
                     /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     5608..5627
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5846..5950)
                     /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 aaaccggagg ttcaggttca ggtggaggat ccggtaataa ggaagcttgc
      961 aaagttttag ccaaacaact tgtgcatcta cggaaacaga agacgagaac ttttgctgta
     1021 agttcaaaag ttacttctat gtctacacaa acaaaagtga tgaattccca aatgaagatg
     1081 gctggagcaa tgtctaccac agcaaaaaca atgcaggcag ttaacaagaa gatggatcca
     1141 caaaagacat tacaaacaat gcagaatttc cagaaggaaa acatgaaaat ggaaatgact
     1201 gaagaaatga tcaatgatac acttgatgac atctttgacg gttctgatga cgaagaagaa
     1261 agccaggata ttgtgaatca agttcttgat gaaattggaa ttgaaatttc tggaaagatg
     1321 gccaaagctc catcagctgc tcgaagctta ccatctgcct ctacttcaaa ggctacaatc
     1381 tcagatgaag agattgaacg gcaactcaag gctttaggag tagatggagg atcctaccca
     1441 tacgacgtcc cagactacgc tggatacccg tatgatgttc cggattacgc tggctaccca
     1501 tatgacgttc cggattacgc ttgagggccc tattctatag tgtcacctaa atgctagagc
     1561 tcgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc
     1621 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga
     1681 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
     1741 cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat
     1801 ggcttctgag gcggaaagaa ccagctgggg ctctaggggg tatccccacg cgccctgtag
     1861 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     1921 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     1981 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     2041 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     2101 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     2161 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     2221 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattaatt
     2281 ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt
     2341 atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca
     2401 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta
     2461 actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga
     2521 ctaatttttt ttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag
     2581 tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctcccggg agcttgtata
     2641 tccattttcg gatctgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat
     2701 ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca
     2761 caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg
     2821 gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcagga cgaggcagcg
     2881 cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact
     2941 gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct
     3001 caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg
     3061 cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt
     3121 actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc
     3181 gcgccagccg aactgttcgc caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc
     3241 gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga
     3301 ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc
     3361 cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt
     3421 atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga
     3481 gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt
     3541 tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg
     3601 gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt
     3661 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     3721 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg
     3781 tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg
     3841 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     3901 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     3961 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     4021 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     4081 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     4141 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     4201 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     4261 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     4321 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     4381 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     4441 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     4501 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     4561 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     4621 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     4681 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     4741 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     4801 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     4861 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     4921 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     4981 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     5041 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     5101 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     5161 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     5221 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     5281 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     5341 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     5401 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     5461 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     5521 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     5581 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     5641 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     5701 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     5761 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     5821 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     5881 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     5941 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt cgacggatcg gga
//
