Cloning vector pURA3-CRISPR, complete sequence

It is a circular DNA molecule with a total length of 9,960 bp. This plasmid was assembled from two commercial plasmids: p414-TEF1p-Cas9-CYC1t (Addgene #43802) provides the Cas9 expression cassette, and p426-SNR52p-gRNA.CAN1.Y-SUP4t (Addgene #43803) provides the gRNA expression elements.

During construction, the URA3 selection marker was retained, and the original SNR52 promoter controlling gRNA expression was replaced with an endogenous tRNAGly promoter. To use this plasmid, only the N20 sequence of the sgRNA needs to be replaced via seamless cloning to target a specific gene. Homology arms of at least 50 bp should be designed at both ends of the donor fragment to ensure homologous recombination efficiency.