Abstract: An efficient synthesis and assembly method for the large fragment DNA based on the programmable nuclease Argonaute, specifically includes: constructing and treating antibiotic resistance gene reconstructed vectors with linearization, dividing a target DNA into multiple small DNA fragments and then synthesizing the small DNA fragments, followed by loading the synthesized small DNA fragments to the antibiotic resistance gene reconstructed vectors; and the SLIC and resistance gene reconstruction are used to achieve assembly of the target DNA. The method combines the SLIC with a resistance gene reconstruction strategy, allowing for the assembly of 5-6 small fragments in a single resistance gene reconstruction, which is more efficient and time-saving. Moreover, the number of the resistance gene reconstructions can be flexibly chosen according to the length of the DNA fragments. Mutations are not introduced caused by PCR, and the reconstructed large fragment do not need a second sequencing, saving time and costs.

Abstract: A Pyrococcus furiosus Argonaute (PfAgo) mutant protein with mesophilic target nucleic acid cleavage activity and an application thereof are provided. The PfAgo mutant protein has an amino acid mutation on at least one of a 617th position and a 618th position relative to the sequence shown in SEQ ID NO: 1, specifically, K617E/G and/or L618Y/F/W/G. Compared with a wild-type PfAgo protein, the provided PfAgo mutant protein has activity in the temperature range of 30-95 Celsius degree (° C.), effectively expanding application range of prokaryotic Argonaute (pAgo) protein.