Fluorescent protein vector for directional selection of PCR clones

Green fluorescent protein (GFP) has become a convenient and versatile tool as a reporter protein in many aspects of science. Here, we show that the en hanced yellow fluorescent protein (EYFP) variant may be used advantageously as a reporter system for directional cloning of blunt-ended PCR products. We have constructed a pUC18-derived plasmid containing a reporter gene codi ng EYFP cloned into the BamHI/HindI-II sites. The blunt-ended PCR product i s cloned into the SmaI site of that plasmid. A reverse PCR primer must be d esigned with extra bases on the 5' end that are required to introduce a rib osome binding site (rbs) for EYFP expression. The reporter gene coding EYFP is not expressed unless an rbs is introduced in the proper orientation at the 3' end of the cloned PCR insert. The results of this cloning procedure may be analyzed by simple visual inspection using a transilluminator In mos t cases, successful directional cloning results in white fluorescent coloni es. The proposed procedure is a convenient method that can reduce the time- and labor-intensive analysis of the clones obtained during blunt-ended PCR product cloning.