Cloning and Modifying ZMYM Genes

Human ZMYM family genes (ZMYM2, ZMYM3, ZMYM4 and ZMYM6) were amplified by PCR from HEK293T cDNAs. Amplified ZMYM gene products were cloned into a pDONR201 vector using Gateway cloning (Invitrogen) following the manufacturer's protocol. ZMYM1 in pENTR223 vector was purchased from Harvard plasmid (Clone ID:HsCD00365611). ZMYM5 in M29 vector was purchased from (GeneCopoeia, Inc., Cat No. EX-Z6750-M29). ZMYM cDNAs were then sub-cloned into pDEST mammalian expression vector as described previously (17 (link)). For ZMYM2, this gene was sub-cloned from pDONR201 into various Gateway destination vectors containing epitope tags as indicated. Deletion mutants of ZMYM2 were created in pDONR201 by PCR-amplifying ZMYM2 cDNA using forward and reverse primers containing complementary sequences to the regions flanking the area to be deleted. SUMO-interacting motif mutant forms of ZMYM2 were generated by site-directed mutagenesis (New England Biolabs) using primers that created alanine substitutions at the indicated amino acid positions.