LOCUS       sequence-462156-        5476 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bicistronic expression of CHMP2B along with an mTagBFP2 transfection
            marker, connected via 2A sequences..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5476)
  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 5476)
  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 mTagBFP2-2A-CHMP2B
FEATURES             Location/Qualifiers
     source          1..5476
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        119..422
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        423..626
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     577..597
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      665..674
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             671..1381
                     /codon_start=1
                     /product="enhanced monomeric blue fluorescent protein 
                     (Subach et al., 2011)"
                     /label=mTagBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRND
                     MALKLVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAV
                     ARYCDLPSKLGHKLN"
     CDS             1391..1444
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             1463..1519
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     polyA_signal    2322..2443
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2359..2378)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2413..2432
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2450..2905)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2587..2608)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2799..2818
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2932..3036
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3038..3395
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3038..3229
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3061..3081)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3246..3381
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3430..4224
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             3430..4224
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3484..3503)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4094..4113
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4412..4431)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4456..4503
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4832..5420
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5321..5340
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg ccatgcatta
       61 gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg
      121 ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga
      181 cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat
      241 gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa
      301 gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca
      361 tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc gctattacca
      421 tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat
      481 ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg
      541 actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac
      601 ggtgggaggt ctatataagc agagctggtt tagtgaaccg tcagatccgc tagcgctacc
      661 ggtcgccacc atggtgtcta agggcgaaga gctgattaag gagaacatgc acatgaagct
      721 gtacatggag ggcaccgtgg acaaccatca cttcaagtgc acatccgagg gcgaaggcaa
      781 gccctacgag ggcacccaga ccatgagaat caaggtggtc gagggcggcc ctctcccctt
      841 cgccttcgac atcctggcta ctagcttcct ctacggcagc aagaccttca tcaaccacac
      901 ccagggcatc cccgacttct tcaagcagtc cttccctgag ggcttcacat gggagagagt
      961 caccacatac gaagacgggg gcgtgctgac cgctacccag gacaccagcc tccaggacgg
     1021 ctgcctcatc tacaacgtca agatcagagg ggtgaacttc acatccaacg gccctgtgat
     1081 gcagaagaaa acactcggct gggaggcctt caccgagacg ctgtaccccg ctgacggcgg
     1141 cctggaaggc agaaacgaca tggccctgaa gctcgtgggc gggagccatc tgatcgcaaa
     1201 cgccaagacc acatatagat ccaagaaacc cgctaagaac ctcaagatgc ctggcgtcta
     1261 ctatgtggac tacagactgg aaagaatcaa ggaggccaac aacgagacct acgtcgagca
     1321 gcacgaggtg gcagtggcca gatactgcga cctccctagc aaactggggc acaagcttaa
     1381 tggctccggc gagggcaggg gaagtcttct aacatgcggg gacgtggagg aaaatcccgg
     1441 cccaggatcc ggagggtctg gagctactaa cttcagcctg ctgaagcagg ctggcgacgt
     1501 ggaggagaac cctggaccta tggcgtccct cttcaagaag aaaaccgtgg atgatgtaat
     1561 aaaggaacag aatcgagagt tacgaggtac acagagggct ataatcagag atcgagcagc
     1621 tttagagaaa caagaaaaac agctggaatt agaaattaag aaaatggcca agattggtaa
     1681 taaggaagct tgcaaagttt tagccaaaca acttgtgcat ctacggaaac agaagacgag
     1741 aacttttgct gtaagttcaa aagttacttc tatgtctaca caaacaaaag tgatgaattc
     1801 ccaaatgaag atggctggag caatgtctac cacagcaaaa acaatgcagg cagttaacaa
     1861 gaagatggat ccacaaaaga cattacaaac aatgcagaat ttccagaagg aaaacatgaa
     1921 aatggaaatg actgaagaaa tgatcaatga tacacttgat gacatctttg acggttctga
     1981 tgacgaagaa gaaagccagg atattgtgaa tcaagttctt gatgaaattg gaattgaaat
     2041 ttctggaaag atggccaaag ctccatcagc tgctcgaagc ttaccatctg cctctacttc
     2101 aaaggctaca atctcagatg aagagattga acggcaactc aaggctttag gagtagatta
     2161 agaattctgc agtcgacggt accgcgggcc cgggatccac cggatctaga taactgatca
     2221 taatcagcca taccacattt gtagaggttt tacttgcttt aaaaaacctc ccacacctcc
     2281 ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt taacttgttt attgcagctt
     2341 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     2401 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttaacgcgta aattgtaagc
     2461 gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa
     2521 taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt
     2581 gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg
     2641 cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt
     2701 ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga
     2761 gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg
     2821 ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg
     2881 cttaatgcgc cgctacaggg cgcgtcaggt ggcacttttc ggggaaatgt gcgcggaacc
     2941 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     3001 tgataaatgc ttcaataata ttgaaaaagg aagagtcctg aggcggaaag aaccagctgt
     3061 ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     3121 aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag
     3181 gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc
     3241 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa
     3301 ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt
     3361 gaggaggctt ttttggaggc ctaggctttt gcaaagatcg atcaagagac aggatgagga
     3421 tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag
     3481 aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc
     3541 cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg
     3601 aatgaactgc aagacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc
     3661 gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg
     3721 ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct
     3781 gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg
     3841 aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat
     3901 ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgagc
     3961 atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg
     4021 gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc
     4081 tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct
     4141 gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat
     4201 cgccttcttg acgagttctt ctgagcggga ctctggggtt cgaaatgacc gaccaagcga
     4261 cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct
     4321 tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg
     4381 agttcttcgc ccaccctagg gggaggctaa ctgaaacacg gaaggagaca ataccggaag
     4441 gaacccgcgc tatgacggca ataaaaagac agaataaaac gcacggtgtt gggtcgtttg
     4501 ttcataaacg cggggttcgg tcccagggct ggcactctgt cgatacccca ccgagacccc
     4561 attggggcca atacgcccgc gtttcttcct tttccccacc ccacccccca agttcgggtg
     4621 aaggcccagg gctcgcagcc aacgtcgggg cggcaggccc tgccatagcc tcaggttact
     4681 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     4741 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     4801 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     4861 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     4921 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     4981 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     5041 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     5101 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     5161 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     5221 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     5281 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     5341 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     5401 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     5461 gctggccttt tgctca
//
