A Method for Seamless Stack Enzymatic Assembly of DNA Molecules Using IIS Type Restriction Endonuclease
Enzyme digestion-enzyme linked is a traditional method for molecular assembly,however,it's appli-cation is restricted by low assembly efficiency,few restriction sites,easy to produce mutant sites and unable to achieve seamless cloning.In order to overcome the above limitations,we propose a Seamless Stack Enzymatic Assembly using IIS type restriction endonuclease.This method is a binary enzyme assembly method which uses IIS type restriction endonuclease recognition site outside the cutting site,and does not destroy the recognition site in the process of linearizing plasmid carrier and homologous recombination with the target fragment,and realizes the seamless cloning of the target sequence by multi round enzyme assembly.And we verified the feasibility and practicability of this method by assembling the CDs sequence of EAT1 gene.
Seamless stackEnzymatic assemblyIIS type restriction endonuclease
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