cDNA cloning

Rat Magi2 (S-SCAM) full-length cDNA was gifted by Dr. Min Loo, Yonsei University College of Medicine, Korea. Human TNS2 isoform 2 full-length cDNA was subcloned by PCR from human full-length cDNA (cDNA clone MGC: 165058, IMAGE: 40148847). Mouse TNS2 was subcloned from mouse TNS2 full-length ORF cDNA (Cat No: MR211954, Origene). Human DLC1 subcloned from human fetal brain cDNA library (Clontech). Human CDK20 full-length was subcloned from human full-length cDNA (cDNA clone MGC: 3757, IMAGE: 3605918). Mouse Cdk20 was subcloned from mouse full-length Cdk20 (cDNA clone, MGC: 38901, IMAGE: 5362198). Human ITSN1 full-length was subcloned from human full-length cDNA (cDNA clone MGC: 134949 IMAGE: 40073781). Expression vectors were produced using LR clonase (Invitrogen®) following the manufacturer’s instruction. The coding sequences of human ITSN2-L was purchased from Origene, and the ITSN2-L with the N-terminal FLAG tag (FLAG-ITSN2-L) or FLAG (mock) was subcloned into pCAGEN using the In-Fusion® HD Cloning Kit (TaKaRa, Clontech). The following expression vectors were used in this publication: pRK5-N-Myc, pCDNA6.2-N-GFP, pCMV6-AN-DDK (Origene) or pCAGEN. pCAGEN was a gift from Connie Cepko (Addgene plasmid # 11160)⁴⁷. Clones reflecting the mutations identified in individuals with NS were introduced in the cDNA constructed by using the Quick change II XL site-directed mutagenesis kit, Agilent Technologies or by the PrimeSTAR® Mutagenesis Basal Kit (TaKaRa). Final sequences were confirmed by sequencing. pRK5-Myc-Cdc42-WT was a gift from Gary Bokoch (Addgene plasmid # 12972).