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Fig. 4 Multisite Gateway approach to generate C-terminal fusion proteins in a pCS-based vector. A: Generation of the pENTR-egfp3 plasmid by BP recombination between an attB1/attB2 flanked PCR product and pDONR221. The reverse attB2 primer was engineered to eliminate the stop codon in the Egfp coding sequence. B: Generation of a C-terminal mCherry tag (p3Emcherry) by BP cloning. In this case, the forward primer contains an attB2 site and the reverse primer an attB3 site. Recombination of the attB2-attB3 PCR product with pDONRP2r-P3 results in an ENTRY clone in which the mcherry coding sequence is flanked by attR2 and attL3. C: Depiction of the two-way multisite Gateway LR reaction between pENTRegfp3, p3Emcherry and pCSDest2. Recombination can only occur between corresponding attL and attR sites resulting in the proper orientation of egfp and mcherry ORFs in pCSegfp-mcherry. Note that att sites are not to scale. D-F: A 24 hours postfertilization embryo injected with 100 pg of egfp-mcherry mRNA synthesized from the plasmid generated in C; lateral view, dorsal is up, anterior to the left; all pictures are from the same embryo. D: Transmitted light illumination.E: Illumination to visualize green fluorescence.F: Illumination to visualize red fluorescence.