SacI, KpnI, SmaI, BamHI, XbaI, SalI, and PstI). Meanwhile, a series of applicative binary expression vectors that can be utilized for subcellular localization were constructed by fusion of the MCS and eGFP. Subsequently, the recombinant vectors were successfully transferred into Arabidopsis thaliana and Nicotiana benthamiana for further investigation of functional elements in these plant binary expression vectors. Our results demonstrated that this system was a convenient and versatile vector system for phenotypic, functional, subcellular localization, and promoter activity analysis, and it provided a relatively high-efficiency and reliable platform for researchers in vector construction and may facilitate large-scale functional genomics analysis in plants.