Ultra-Universal One Step SeamLess Cloning Mix is a simple, fast and efficient seamless cloning kit developed based on the principle of homologous recombination. The kit does not rely on tedious enzyme digestion and ligating steps and allows for directional cloning of DNA into any site of any vector without restriction enzyme sites limitations. Linearize the vector completely and introduce 15-25 bp homologous sequences of the ends of the linearized vector to the 5' end of the forward and reverse PCR primers of the insert, mix the PCR products, both ends of which contain sequences identical to the vector ends, with the linearized vector in a certain ratio and perform transformation under the reaction of 2×Ultra Cloning MasterMix at 50 ℃ for 5-30 minutes. This process allows for directional cloning of 1-5 fragments with a positivity rate of over 95%. Unique cofactors and stabilizers have been added to 2×Ultra Cloning MasterMix to effectively improve the efficiency of recombination. The optimized reaction Buffer system has better compatibility and better guarantees the positive cloning rate of the complex system.