Mouse Ccnb3 mRNA was amplified by PCR from a whole-testis cDNA library and cloned into pRN3-RFP vectors using the In-Fusion cloning kit (Clontech). X. laevis cyclin B3 cDNA (clone ID: 781186; NCBI ID: 379048) was purchased from Dharmacon and used as a template for PCR amplification. D. rerio and D. melanogaster cyclin B3 (NCBI ID: 767751 and 42971, respectively) were amplified by PCR from cDNA from D. rerio embryo or the S2 cell line, respectively. Amplified products for X. laevis, D. rerio, and D. melanogaster were cloned into pRN3-GFP vector using the In-Fusion cloning kit (Clontech). To generate Ccnb3 MRL mutation, overlapping primers with specific mutation were used to amplify PCR product from the wild-type template pRN3–cyclin B3–RFP, the template was digested by DpnI treatment, and the PCR product was used to transform Escherichia coli DH5α competent cells. To generate the ΔD-box mutation, PCR primers that delete the D-box were used to amplify the Ccnb3 plasmid, and the resulting PCR product was phosphorylated and ligated. The primer list can be found as Table S1. Plasmids for in vitro transcription of cyclin B1–GFP, securin-YFP, histone H2B-RFP, cyclin A2–GFP, and β-tubulin-GFP were used (Brunet et al., 1998 (link); Herbert et al., 2003 (link); Terret et al., 2003 (link); Tsurumi et al., 2004 (link); Touati et al., 2012 (link)).