Seamless cloning and assembly technique is a new, rapid and concise cloning method, which aims to overcome the above defects. It can insert one or more fragments of target DNA at any site of the plasmid, without any restriction enzyme and ligase. The recombinant vector with high efficiency can be obtained by a one-step recombination method, which breaks through the traditional double enzyme digestion and linking. 1. Flexible loci selection: gene cloning at any position of the vector; 2. Fast and simple: the process of enzyme cutting, rubber cutting recovery and enzyme bonding was omitted, and the carrier construction was completed in about 1 hour; 3. Precision: there is no need to add any additional procedures; 4, high cloning efficiency, positive clones up to 90%; 5. Recombination of multiple fragments of target genes at a time.