Genetically Engineered Protein Constructs

TurboID was a kind gift of A. Ting (Addgene #107171)^{29 (link)}. PMLIVa^WT and PMLIVa^3MAS were previously described^{21 (link)}. SUMO1, SUMO2, Ub, RANGAP1 and UBC9 ORFs were amplified from U2OS cell cDNA by high-fidelity PCR (Platinum SuperFi DNA Polymerase; Invitrogen). A GSQ linker (GGGSSGGGQISYASRG) was placed between the C-terminal part of TurboID and the UbLs as well as between the N-terminal part and the substrates. All constructs were generated by standard cloning or by Gibson Assembly (NEBuilder HiFi Assembly, NEB) using XL10-Gold bacteria (Agilent). Depending on the construction, plasmid backbones derived from EYFP-N1 (Clontech/Takara), Lenti-Cas9-blast (a kind gift of F. Zhang; Addgene #52962) or TRIPZ (Open Biosystems/Horizon) were used. After assembly, all vectors were validated by sequencing. Additional details for constructs are described in Supplementary Table 1. Oligonucleotides sequences are shown in Supplementary Table 2. The sequences of representative constructs are in the Source Data file. Cloning details about other constructs are available upon request.