The study investigates the cleavage efficiency of four different 2A peptides (P2A, T2A, E2A, and F2A) in various contexts, including human cell lines, zebrafish embryos, and adult mice. The 2A peptides are derived from different viruses, such as foot-and-mouth disease virus, equine rhinitis A virus, *Thiose asigna* virus, and porcine teschovirus-1. The researchers generated expression plasmids harboring these 2A peptides and evaluated their cleavage efficiency using Western blotting and confocal microscopy. The results showed that the P2A peptide had the highest cleavage efficiency in all tested contexts, followed by T2A, E2A, and F2A. The study also constructed cloning vectors with a simian cytomegalovirus (scMV) promoter and multiple cloning sites to facilitate the use of 2A technology for coexpression of multiple genes. The findings suggest that the P2A peptide is a highly efficient tool for bicistronic or multicistronic expression in biomedical research.The study investigates the cleavage efficiency of four different 2A peptides (P2A, T2A, E2A, and F2A) in various contexts, including human cell lines, zebrafish embryos, and adult mice. The 2A peptides are derived from different viruses, such as foot-and-mouth disease virus, equine rhinitis A virus, *Thiose asigna* virus, and porcine teschovirus-1. The researchers generated expression plasmids harboring these 2A peptides and evaluated their cleavage efficiency using Western blotting and confocal microscopy. The results showed that the P2A peptide had the highest cleavage efficiency in all tested contexts, followed by T2A, E2A, and F2A. The study also constructed cloning vectors with a simian cytomegalovirus (scMV) promoter and multiple cloning sites to facilitate the use of 2A technology for coexpression of multiple genes. The findings suggest that the P2A peptide is a highly efficient tool for bicistronic or multicistronic expression in biomedical research.