Pcmv sport plasmid

The full-length human MISP sequence harbored in a pCMV-SPORT plasmid (Harvard PlasmID Database; HsCD00326629) was subcloned by PCR and TOPO-cloned into a pCR™8 Gateway entry vector (Invitrogen; 46–0899). In-frame sequence insertion was confirmed by sequencing. MISP was then shuttled into Gateway-adapted plasmids: pEGFP-C1, pmCherry-C1, and pHALO-C1. Similarly, the human beta-actin and UtrCH sequences were cloned and shuttled into a Gateway adapted HALO-C1 plasmid. To create lentiviral expression vectors, the human MISP sequence was subcloned by PCR and inserted into a puromycin-resistant pLVX1-EGFP backbone by restriction enzyme digestion using XhoI and BamHI. The human fimbrin and villin sequences were cloned into a pEGFP-C1 plasmid (Clontech; 6084–1). The pEGFP-N1 construct harboring the human ezrin sequence was purchased from Addgene, plasmid# 20680. The pEGFP-C1-espin (rat small espin) was a generous gift from Dr. Jim Bartles. To create baculovirus expression vectors, the MISP and EGFP-MISP sequences were subcloned into modified pFastBac-6xHis-MBP LIC expression vector (Addgene; plasmid #30116). All constructs were confirmed by sequencing.