The pGW-HA.attB plasmid is designed for generating donor plasmids that can be used to insert sequence containing an open reading frame (ORF) of interest, expressed under the control of UASt regulatory sequences and tagged at the C-terminal end with three copies of the Tag:HA epitope, into an attP-containing docking site in the genome, via phiC31:int-mediated integration. The ORF is flanked by a pair of incompatible FRT sites (FRT5 at the N-terminal end and FRT2 at the C-terminal end, upstream of the Tag:HA sequence), which allows for future in vivo exchange of either the promoter or the C-terminal tag sequence. A 24 residue flexible linker separates the FRT2 and Tag:HA sequences, to minimize potential steric interference between the protein encoded by the ORF of interest and the tag. A loxP site is present upstream of the UASt sequences. An ORF of interest can be inserted into the plasmid via a Gateway cassette.
