Recombinant-based seamless cloning technology offers a simple, fast, and efficient method for DNA directional cloning without the need for laborious enzymatic digestion, ligation steps, or end-blunting procedures. This technique relies on the recombination of DNA fragments with 15 to 25 nucleotides of homologous sequences at the ends of linearized vectors. It allows the cloning of insert fragments into any desired position of a linear vector, with minimal self-ligation background. The LightNing® DNA Assembly Mix Plus seamless cloning kit enables the recombination of single or multiple DNA fragments in a single reaction. It can achieve single fragment assembly in as little as 5 minutes, with a positive clone rate exceeding 95%. The mix contains auxiliary factors that effectively enhance the cloning efficiency, and the optimized reaction system tolerates impurities present in unpurified PCR products to some extent. The upgraded version of the seamless cloning kit offers higher positive clone rates and improved compatibility.