Generation of Engineered mGluR2 Constructs

The C-terminal FLAG-tagged mouse mGluR2 construct in pcDNA3.1(+) expression vector was purchased from GenScript (ORF clone: OMu19627D) and verified by sequencing (ACGT Inc). Full length mGluR2 construct with an amber codon (TAG) mutation of amino acid A548 (azi-CRD) or N-terminal SNAP-tag (SNAP-mGluR2) were generated as previously reported (Liauw et al., 2021 (link)). The insertion of an amber codon (TAG) between E715 and V716 in mGluR2 (azi-ECL2) was performed using the QuikChange site-directed mutagenesis kit (Agilent). SNAP-mGluR2 constructs used for calcium imaging had C-terminal FLAG-tag removed by PCR-based deletion using phosphorylated primers. All plasmids were sequence verified (ACGT Inc). DNA restriction enzymes, DNA polymerase and DNA ligase were from New England Biolabs. Plasmid preparation kits were purchased from Macherey-Nagel.

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Liauw B.W., Foroutan A., Schamber M.R., Lu W., Samareh Afsari H, & Vafabakhsh R. (2022). Conformational fingerprinting of allosteric modulators in metabotropic glutamate receptor 2. eLife, 11, e78982.

Publication 2022

pcDNA3.1(+) expression vector ORF clone: OMu19627D QuikChange site-directed mutagenesis kit DNA polymerase DNA ligase Plasmid preparation kits

Amber codon Amino acid Calcium Clone Deletion Dna ligase Dna polymerase Dna restriction enzymes Mglur2 Mouse Mutation Plasmid Primers Site directed mutagenesis Vector

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Variable analysis

independent variables

Insertion of an amber codon (TAG) between E715 and V716 in mGluR2 (azi-ECL2)

dependent variables

Not explicitly mentioned

control variables

C-terminal FLAG-tagged mouse mGluR2 construct in pcDNA3.1(+) expression vector
Full length mGluR2 construct with an amber codon (TAG) mutation of amino acid A548 (azi-CRD)
N-terminal SNAP-tag (SNAP-mGluR2) constructs
SNAP-mGluR2 constructs used for calcium imaging had C-terminal FLAG-tag removed by PCR-based deletion using phosphorylated primers

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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