Quikchange primer design program

Site-directed mutants of the A_2AAR were generated by polymerase chain reaction (PCR) mutagenesis as described previously^{29 (link)}. pcDNA3.1(+)-hA_2AAR with N-terminal HA and FLAG tags and a C-terminal His tag was used as the template. Primers for mutants L249V^6.51 and L249A^6.51 were designed by the QuikChange Primer Design Program of Agilent Technologies (Santa Clara, CA, USA) and primers were obtained from Eurogentec (Maastricht, The Netherlands). All DNA sequences were verified by Sanger sequencing at LGTC (Leiden, The Netherlands).

SARS-CoV-2 nsp7, nsp8, nsp12, nsp13, nsp14 and nsp16 coding sequences (CDSs) were codon-optimized for bacterial expression and synthesized as gBlocks (Integrative DNA Technologies). The CDSs for nsp9 and nsp10 were amplified from mammalian expression vectors (a gift from N. Krogan)^{40 (link)}. The CDSs were cloned into modified pET28a bacterial expression vectors containing N-terminal 6/8/10×His tags followed by the yeast Sumo (smt3) CDS amino acid mutations were introduced using QuikChange site-directed mutagenesis. In brief, primers were designed using the Agilent QuikChange primer design program to generate the desired mutation and were used in PCR reactions with PfuTurbo DNA polymerase. The reaction products were digested with Dpn1, transformed into DH5α cells and mutations were confirmed by Sanger sequencing.
For protein expression in Escherichia coli, ppSumo-SARS-CoV-2 nsps and mutants were cloned into a BamH1 site at the 5′ end, which introduced a Ser residue following the diGly motif in smt3. To make native N termini, the codon encoding the Ser was deleted using QuickChange mutagenesis. Thus, after cleavage with the ULP protease (after the diGly motif), the proteins contained native N termini. pGEX-2T-GST-eIF4E K119A^{41 (link)} was obtained from Addgene (112818).

cDNAs of human FBX O 38 (Origene #RC204380), ZXDA (TransOMIC Technologies, #BC059356), or ZXDC (kind gift from Dr. Joseph D. Fontes.) genes were cloned into pcDNA3.1 containing either N-terminal twinStrepII-FLAG-tag (pcDNA3.1—NSF) or an N-terminal hemagglutinin-tag (pcDNA 3.1—HA). For mutated and truncated variants of these genes, PCR mutagenesis was carried out using primers designed with QuikChange^® Primer Design Program (Agilent) with subsequent DpnI digestion. All the constructs created were verified by sequencing. For inducible expression, cDNAs of selected genes were cloned into lentiviral pTRIPZ™ (Dharmacon) or pSBtet vectors. A retroviral construct expressing eGFP-CENPB was a gift from Iain Cheeseman (pKG141-eGFP-CENPB; Addgene plasmid #69759). The cDNA library of the ubiquitin ligases was prepared as described previously (Lidak et al., 2021 (link)).
For FBXO38^{∆Ex3/∆Ex3} cell line generation (HCT116 and RPE-1 FBXO38 KO), single guide RNAs (sgRNAs) were cloned into the LentiCRISPR (pXPR_001; Addgene, #49535) containing dual sgRNA and hSpCas9 expression cassettes. Optimal sgRNA sequences were designed using http://crispr.mit.edu/and the targeted sequences were: AAG​CTG​TAT​GAC​CGT​ATG​TGT​GG, GAT​GCA​TGT​TTT​CCG​GTG​AAT​GG. Successful deletion was confirmed by PCR and sequencing.

The donor protein in bioluminescence resonance energy transfer (BRET) assay, mouse SCTR tagged at the carboxyl-terminus with Renilla luciferase construct (mSCTR-Rlu) was the generous gift of Prof. L.J. Miller from previous study [35 (link)]. Mouse AVPR 1a, 1b and 2, as well as β-arrestin (mAVPR1a, mAVPR1b, and mAVPR2; β-arrestin) were tagged at carboxyl end with yellow fluorescent protein (YFP) by cloning the respective cDNA into the vector pEYFP-N1 (Promega, Fitchburg, WI) as acceptor proteins in BRET (mAVPR1a/AVPR1b/AVPR2-YFP). Untagged mSCTR, mAVPR1a, mAVPR1b, and mAVPR2 were cloned into the vector pcDNA3.1(+). mAVPR2 mAVPR2 mutants were generated by site-directed mutagenesis using the QuikChange II Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA). The primers were designed using the online QuikChange Primer Design program (Agilent Technologies). The Rlu-tagged AVPR2 mutants were obtained by cloning the receptor into the Rlu-plasmid.