POSITIVE SCREENING METHOD IN YEAST FOR THE SYNTHETIC GENES WITHOUT FRAMESHIFT AND NONSENSE MUTATIONS

摘要

The present invention is intended to selectively select clones that do not contain an unintended transcriptional modification base error, i.e., base deletion and insertion and termination codon mutation, in gene synthesis for protein expression.To this end, a synthetic gene was inserted between the DNA binding site (LexA DBD, LexA DNA Binding Domain) of the E. coli Lexa protein present in the E. coli-yeast shuttle vector prepared in the present invention and VP16, In vivo cloning of yeast without the use of restriction enzymes and ligases, VP16 is generated only when there is a gene that does not have a transcriptional and terminator mutation. Thus, an operator that binds to LexA DBD in the host yeast And HIS3 and ADE2 repressor genes including histidine and adenine auxotrophic host yeast can be grown on minimal medium to selectively clone the synthetic gene and select genes without the transcriptional and termination codon mutations Method.;