Construction of plasmids and bacmids

All the plasmids used in this research for transient expression were prepared by standard molecular cloning protocols. The indicated genes, gene truncations, and genes with epitope tags were generated by PCR or site-directed mutagenesis (Transgene) and inserted into pIZ-V₅/Ha vectors (Invitrogen).

To prepare recombinant bacmids, the Bac-to-Bac system was employed according to Invitrogen’s protocol. In brief, Ac34 expression cassettes controlled by the native ac34 promoter were cloned into pFbdg, a pFastbac-Dual vector (Invitrogen) bearing an EGFP expression cassette controlled by the p10 promoter [31]. The resulting shuttle vectors were then used to transform DH10B E. coli cells harboring the vAc34KO bacmid provided by Cai et al. to generate the transposed bacmid constructs [36].

Maps of the plasmids and bacmids prepared in this research are diagramed in S1 Fig.