Generation of Chimeric KLC1/2 Constructs

pCIneo-Flag-KLC1A (pFlag-KLC1) and pCIneo-Flag-KLC2 (pFlag-KLC2) have been described [52 (link),71 (link)]. These plasmids express N-terminal Flag-tagged full length versions of murine kinesin-1 light chain isoform 1A (accession number NM_008450.2) and 2 (accession number NM_008451.2) respectively.
Chimeric KLC1/2 alleles were generated by splicing by overlap extension [72 (link)] (using the combination of primers listed in S1 and S2 Tables). Briefly the 5’ and 3’ fragments were generated by PCR using a high fidelity DNA polymerase (platinum Pfx, Invitrogen) with short complementary overlapping regions. These were spliced together and amplified using overlapping PCR and cloned into the EcoRI and XbaI sites of pCI-neo (Promega).
An E2 open reading frame (ORF, codon optimised for expression in human cells, GeneArt), was subcloned into the NotI-XbaI restriction site of pcDNA3-HA and pcDNA3-V5. These plasmids are two pcDNA3 (Invitrogen) variants containing the coding sequence for either an N-terminal HA-epitope tag with alanine linker (MYPYDVPDYAAAA) or a V5-epitope tag with alanine linker (MGKPIPNPLLGLDSTAAA) inserted into the EcoRI-NotI site.