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Molecular cloning methods based on primer and overlap extension PCR are widely used due to their simplicity, reliability,low cost and high efficiency. In this article, an efficient mega primer (MP)-mediated cloning strategy for chimeragenesis and long DNA fragment insertion is presented. MP cloning is a seamless, restriction/ligation-independent method that requires only three steps: i) the first PCR for mega primer generation; ii) the second PCR for exponential amplification mediated by the mega primers; and iii) DpnI digestion and transformation. Most importantly, for chimeragenesis, genes can be assembled and constructed into the plasmid vector in a single PCR step. By employing this strategy, we successfully inserted four DNA fragments (approximately 500 bp each) into the same vector simultaneously. In conclusion, the strategy proved to be a simple and efficient tool for seamless cloning. A C C E P T E D M A N U S C R IP T 10.1042/BSR20160608 . Please cite using the DOI 10.1042/BSR20160608 http://dx.doi.org/ up-to-date version is available at encouraged to use the Version of Record that, when published, will replace this version. The most this is an Accepted Manuscript, not the final Version of Record. You are : Bioscience Reports