Here, we improved the OEP by supplying primers during OEP, which allows exponential amplification of OEP products. The promising exonuclease-based methods utilize enzymes with exonuclease activity to generate single-stranded DNA ends for complementary annealing 11, 12, 13, 14, 15, 16, 17, 18, which mimic in vivo recombination. Specific strains are required for in vivo recombination to enhance its efficiency 10. However, due to the inefficient priming of megaprimer, OEP can be used only for inserts less than 6.7 kilobases (kb) 7. Numerous restriction-free cloning techniques, including overlap extension PCR (OEP) methods 3, 4, 5, 6, 7, 8, in vivo recombination 9, 10 and exonuclease-based methods 11, 12, 13, 14, 15, 16, 17, 18, have been developed to overcome the site limitation of restriction endonuclease. The classical gene cloning requires the vector and the target fragment have compatible restriction endonuclease cleavage sites, thus the cloning site of a target gene is often limited 1, 2.