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Construction of the Grasp65 phosphorylation-negative mutant. Grasp65wt was cloned into pEGFP-N1 to generate Grasp65-GFP. Overlapping mutagenic primers were used to generate alanine mutations at serines and threonines located in mitotic kinase target sequences in the S/P domain. The resulting mutant was designated Grasp65-7A. To generate lentiviral constructs, wt and mutant Grasp65-7A–GFP constructs were subcloned into the pLVX-puro vector.
