Shuttle vectors for direct in frame cloning and construction of gfp and gus/gfp reporter gene fusions

Abstract

Two shuttle plasmid vectors have been constructed that facilitate the direct in frame cloning upstream of gfp and gus/gfp reporter genes. Coding sequences of gfp and gus/gfp reporter genes were inserted into multicloning site of pUC19 cloning vector in a way to create frame shift and prevent gfp and gus/gfp expression from the lac promoter. Blunt end PCR amplified fragments, corresponding to signal peptide or other translated gene regions, were cloned into Sma I site upstream of gfp and gus/gfp genes. The recombinant colonies, containing correct in frame fusion, were directly selected after appearance of green (GFP) fluorescence under UV light. The use of the developed cloning vectors for construction of gene fusions and plant transformation vectors are discussed. © 2004 Taylor and Francis Group, LLC.