Plvsin cmv puro

A cDNA fragment encoding mouse Ulk1 was amplified from the genomes of wild-type MEFs with forward (5′-AAGGGATCCGAATTCATGGAGCCGGGCCGCGGCGGCG-3′) and reverse (5′-AGATGCATGCTCGAGTCAGGCATAGACACCACTCAGCAG-3′) primers. It was cloned into EGFP-pcDNA at EcoRI-XhoI sites, resulting in GFP-Ulk1-pcDNA. A cDNA for GFP-Ulk1 was amplified from GFP-Ulk1-pcDNA with forward (5′-ATTTCCGGTGAATTCATGGTGAGCAAGGGCGAG-3′) and reverse (5′-GGTAGAATTGGATCCTCAGGCATAGACACCACTCAGCAG-3′) primers and was cloned into pLVSIN-CMV-puro (TaKaRa), resulting in GFP-Ulk1-pLVSIN-CMV-puro. To generate pmCherry-p62, p62 was amplified by PCR and digested with EcoRI-XhoI. The PCR product was subcloned into the EcoRI-SalI site of the pmCherry-C2 vector (Clontech, Mountain View, CA).