Principle of constructing vector by homologous recombination

Seamless cloning, also known as homologous recombination, can insert single or multiple fragments into a vector at the same time without repeated digestion and purification. Usually seamless cloning can complete the connection of multiple fragments within 5 15 min, and the positive rate of cloning is as high as 98%. However, it should be noted that when we perform PCR amplification before seamless cloning, there needs to be a homologous sequence of 1520 bases between vectors and fragments, and between fragments. Why? Because there are three enzymes in our seamless cloning kit, which are T5 nucleic acid exonuclease, DNA polymerase and DNA ligase. Among them, the nucleic acid exonuclease of T5 nucleic acid has the nucleic acid exonuclease activity of 5'3' nucleic acid and can degrade the sequence starting from 5'3'. At this point, the 3' homologous sequences of the vector and fragment are exposed. After base complementary pairing, DNA polymerase adds the missing base, and DNA ligase connects the fragment with the vector, thus realizing the function of vector recombination. Therefore, if homologous sequences, vectors and fragments are not added, fragments and fragments will not be connected.