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pcDNA3.1 V5 His-TOPO-GluCl optbeta-m-YFP Y182F RSR_AAA (Opt β-Gluclv2.0)
(Plasmid #47542)

Ordering

Item Catalog # Description Quantity Price (USD)
Plasmid 47542 Standard format: Plasmid sent in bacteria as agar stab 1 $75

This material is available to academics and nonprofits only.

Backbone

  • Vector backbone
    pcDNA3.1/V5-His TOPO
  • Backbone manufacturer
    Invitrogen
  • Backbone size w/o insert (bp) 2037
  • Total vector size (bp) 5523
  • Modifications to backbone
    Codon optimized sequences of the C. elegans GluCl channel, including optGluCl WT, optGluCl WT, optGluCl -XFP, and optGluCl -XFP (4), cloned into plasmid vector pcDNA3.1/V5-His TOPO (catalog no. K4800-01, Invitrogen) were used in this study. Fluorescent protein insertions (XFP) include enhanced yellow (YFP) and cyan (CFP) variants and are located in the M3-M4 loop (3). For convenience, the “opt” nomenclature is omitted throughout most of this paper. Mutations were made using the QuikChange II XL site-directed mutagenesis kit (catalog no. 200522, Agilent Technologies) and PfuTurboDNApolymerase (catalog no. 600250, Agilent Technologies). for the ()RSR_AAA mutation; 5-CG GCC CGC AAG GCC CAG GCA GCC GCC GAG AAG CTG GAG ATG G-3 and 5-C CAT CTC CAG CTT CTC GGC GGC TGC CTG GGCCTTGCGGGCCG-3 for the ()RRR_AAA mutation. A C-terminal V5 tag was added to  and  subunits by mutating the stop codon using the following primers: 5-G CAG AAC GTT CTG TTC GGA GCT AGC AAG GGC AA-3 and 5-TT GCCCTTGCTAGCTCCGAACAGAACGTTCTGC-3 for the  subunit; 5-CC GAG TCC CTG GTG TTG GCT AGC AAG G-3 and 5-C CTT GCT AGC CAA CAC CAG GGA CTC GG-3 for the  subunit
  • Vector type
    Mammalian Expression
  • Selectable markers
    Neomycin (select with G418)

Growth in Bacteria

  • Bacterial Resistance(s)
    Ampicillin
  • Growth Temperature
    37°C
  • Growth Strain(s)
    Top10
  • Copy number
    High Copy

Gene/Insert

  • Gene/Insert name
    Glutamate-gated chloride channels
  • Alt name
    GluCl optbeta-mYFP Y182F RSR_AAA
  • Species
    C. elegans (nematode)
  • Insert Size (bp)
    2037
  • Mutation
    retention motif in ER (starting at R340) changed from RSR to AAA in the intracellular loop of Beta subunit; Y182F; monomerizing A206K in YFP
  • GenBank ID
    AY273892.1
  • Promoter CMV

Cloning Information

Resource Information

How to cite this plasmid ( Back to top)

These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which the plasmids were described, and include Addgene in the Materials and Methods of your future publications.

  • For your Materials & Methods section:

    pcDNA3.1 V5 His-TOPO-GluCl optbeta-m-YFP Y182F RSR_AAA (Opt β-Gluclv2.0) was a gift from Henry Lester (Addgene plasmid # 47542 ; http://n2t.net/addgene:47542 ; RRID:Addgene_47542)
  • For your References section:

    An Engineered GluCl Channel for Sensitive, Consistent Neuronal Silencing by Ivermectin. Frazier SJ, Cohen BN, Lester HA. J Biol Chem. 2013 May 29. 10.1074/jbc.M112.423921 PubMed 23720773