Molecular Cloning of Chimeric Dynein-SRS Constructs

Standard molecular cloning methods were used to create chimeric constructs of monomeric Thermus thermophilus seryl-tRNA synthetase (SRS) fused to the stalk and MTBD of yeast dynein using SRS85:82, a construct with the near full-length mouse stalk and MTBD fused to the coiled-coil base of SRS in the α-registry^{27 (link),33 (link)} as a template. Genomic yeast DNA was amplified to generate DNA fragments of the dynein stalk and MTBD. PCR products were then stitched with partial SRS sequences to enable insertion between the restriction enzyme sites, SalI and PstI, of the original vector (see Supplementary Table 2 for the list of primers used). Single point mutations in the SRS chimeras were generated using the Q5 site-directed mutagenesis kit from NEB. High efficiency 5-α competent E. coli cells (NEB) were used for transformation and plasmid amplification. Single colonies were inoculated in 3 mL of terrific broth (TB) with 30 μg/mL of kanamycin and grown overnight shaking at 37 °C. Plasmids (listed in Supplementary Table 1) were purified using the PureYield plasmid miniprep kit from Promega and verified by standard sequencing.