Cloning procedures

Full-length cDNAs encoding USP7, USP7-C223S, PCGF1-6, and USP7-interacting proteins were cloned into pENTR11 (Invitrogen). Vectors that express enhanced GFP (eGFP)–fusion proteins were generated by recombining coding sequences into destination vectors pCS-eGFP-DEST or pCS-Cherry-DEST using LR Clonase II–mediated recombination. pDEST-3xFLAG was constructed from pCS-eGFP-DEST by replacing the GFP coding sequence with 3xFLAG using the Age I and Bgl II restriction sites. This vector was used to generate the pDEST-3xFLAG-PCeGF1-6 plasmids. All other Flag-tagged constructs were made in either pLX302 or a gateway-compatible version of pQCXIP (Promega; pQCXIP-DEST) after engineering a 3xFLAG-tag in the corresponding pENTR11 clones. CRISPR constructs were made in pSPCas9(BB)-2A-Puro (PX459) V2.0, using the Bbs I restriction sites, or in pLentiCRISPRv2GFP, using Esp3 I restriction sites. The pTRE3GBI_eGFP plasmid was constructed by inserting eGFP using the Eco RI and Kpn I restriction site. The full-length cDNA encoding Ring1A was cloned into the pTRE3GBI_eGFP using Kpn I and Pst I restriction sites. The Ring1A mutants I50S and C72A were produced by site-directed mutagenesis PCR. The RING1B-dTAG construct was made in pLEX_305-N-dTAG (Addgene, plasmid #91797). The integrity of all constructs that we generated was verified by DNA sequencing of the entire coding sequence and flanking regions. See table S1 for an overview of all plasmids.