In vitro Synthesis and Cloning of Fluorescent Protein Plasmids

To generate cRNAs, plasmids were linearized and in vitro transcribed using a mMessage mMachine T3 (Ambion #AM1348) and T7 kits (Ambion #AM1344), according to manufacturer’s protocol. The synthesized cRNAs were then purified using an RNAeasy kit (Qiagen #74104) and stored at -80°C. The pYX-EGFP plasmid was created by transferring T3-T7 cassette from pRNA-EGFP vector [66 (link)] into the pXY-Asc vector (NIH, Bethesda, MD, USA) using PCR cloning. The pYX-EYFP plasmid was created from pYX-EYFP plasmid by replacing coding sequence for EGFP by EYFP. AURKC coding sequence [31 (link)] was cloned by PCR into pYX-EYFP to create pYX-AURKC-EYFP plasmid. pIVT-AURKB/C-EGFP and pGEMHE-mEGFP-mCDK5RAP2 plasmids were described previously [22 (link), 31 (link)].