Developing for almost half a century, plasmid-construction has explored more than 37 methods. Some methods have evolved into new versions. From a global and evolutionary viewpoint, a review will make a clear understand and an easy practice for plasmid-construction. The 37 methods employ three principles as creating single-strand overhang, recombining homology arms, or serving amplified insert as mega-primer, and are classified into three groups as single strand overhang cloning, homologous recombination cloning, and mega-primer cloning. The methods evolve along a route for easy, efficient, or/and seamless cloning. Mechanism of plasmid-construction is primer annealing or/and primer invasion. Scar junction is a must-be faced scientific problem in plasmid-construction.
Keywords: Annealing; Evolution; Mega-primer; Plasmid-construction; Recombination.
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