Oligonucleotide primers

All plasmids and primers used in genetic manipulations are listed in Supplementary Tables 2 and  3, respectively. All basic microbiological and molecular procedures were executed according to standard protocols⁸⁷ . Genomic DNA (gDNA) isolation, plasmid preparation, and DNA gel extraction were performed using nucleotide purification kits purchased from Qiagen or BioBasics. All restriction enzymes, T4 DNA ligase, Quick ligase, Taq DNA polymerase, RNase A, Antarctic phosphatase, and shrimp alkaline phosphatase were purchased from New England Biolabs. Phusion DNA polymerase was purchased from ThermoFisher Scientific. Transformations of P. aeruginosa and P. fluorescens were carried out using established protocols for electroporation^{88 (link)}. Site-directed mutagenesis of plasmids was carried out using the QuikChange II XL Site-Directed Mutagenesis Kit (Agilent). Oligonucleotide primers were purchased from Integrated DNA Technologies. Protein concentrations were measured using the Pierce 660 nm protein assay with the addition of the ionic detergent compatibility reagent (IDCR) when necessary (ThermoFisher Scientific).

Mutagenesis was performed on the cDNA of human GRIN2A in the pCI-Neo vector (pCMV GluN2A). GluN2A mutations with respect to the amino acid numbering in NP_000824.1 were C436R, T531M, R518H, K669N, L812M. Oligonucleotide primers (Integrated DNA Technologies) were designed to incorporate single-base substitutions using the GeneArt^® Site-Directed Mutagenesis Kit (Thermofisher Scientific) (Table S3). The presence of each mutation was confirmed by DNA sequencing (Eurofins Genomics) and the whole coding sequence was also examined to ensure that no other mutations were introduced by polymerase chain reaction (PCR) errors. Plasmid DNA was amplified and purified using EZNA endo-free plasmid maxi kit (VWR International).

Plasmid pET17b-CYVaV, which contains the entire length of WT CYVaV genomic RNA (gRNA) complementary DNA (Accession number JX101610) downstream of a T7 promoter (33 (link)), was used as a template for polymerase chain reaction (PCR)-based site-directed mutagenesis. The desired mutations were introduced using custom-designed oligonucleotide primers (Integrated DNA Technologies) using Q5 high-fidelity DNA polymerase (New England Biolabs). The resulting PCR products were subjected to DpnI digestion followed by T4 DNA ligase before transformation into DH5α Escherichia coli cells. The presence of the desired mutations was confirmed through Sanger sequencing (Eurofins Genomics).