Significance The ability of eukaryotic cells to pass their genomes properly from one cell generation to the next depends on the 1.2-MDa ubiquitin ligase complex APC/C (anaphase-promoting complex/cyclosome) and on the correct timing of its activation by the substrate adaptor CDC20 (cell division cycle 20). Although it has been known for two decades that mitotic APC/C phosphorylation is required for its activation by CDC20, the mechanistic basis of this process remained unknown, in part because the existence of numerous phospho-sites on APC/C made systematic mutagenesis approaches difficult. Here we have used the biGBac technique for the rapid assembly of multigene expression constructs to overcome this limitation and discovered that APC/C contains an autoinhibitory loop region that prevents CDC20 binding until it becomes phosphorylated in mitosis.