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

• Purpose: engineered receptor improves heteromeric receptor expression and sensitivity to IVM.
• Depositing Lab: Henry Lester
• Publication: Frazier et al J Biol Chem. 2013 May 29.

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, 100 μg/mL
• 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

• Cloning method: TOPO Cloning
• 5′ sequencing primer: CMV-F; T7
• 3′ sequencing primer: BGH-Rev

Resource Information

• Article Citing this Plasmid:
  • 1 Reference

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
• Industry Terms:
  • Not Available to Industry