Cloning and Mutagenesis of Human Proteins

The templates for the constructs used were as follows: human spinophilin (REFSEQ: NM_032595.4)—modified from Dr. Maria Vivo (University of Naples “Federico II”); human NF-M (Uniprot ID P07197; Transomics BC096757-seq); human PKAc—pDONR223-PRKACA; human CDK5—pDONR223-CDK5; and human p35—pDONR223-CDK5SR1 were kind gifts from William Hahn & David Root [22 (link)] (plasmid numbers 23495, 23699, and 23779, Addgene, Cambridge, MA). PCR products were generated from these template DNAs and inserted into pDONR221 (ThermoFisher, Waltham MA) for Gateway cloning into either pcDNA3.1/nV5-DEST (NF-M) or modified pcDNA3.1/nV5-DEST vectors containing an HA-tag (spinophilin), myc-tag (PKAc or p35), or FLAG-tag (CDK5) in place of the V5 tag. Mutant spinophilin (Ser17Ala) or PKA (Lys72His) constructs were generated using QuickChange mutagenesis (Agilent Technologies, Santa Clara, CA). PCR was performed with Q5 Hot Start TAQ (New England Biolabs, Ipswich, MA) or VAPRase (Vanderbilt Antibody Protein Resource, Vanderbilt University, Nashville, TN). All constructs and mutations were sequence validated (Genewiz, South Plainfield, NJ).