title: Design 1_Golden Gate_BsaI at Yeast stage_SR2
design: First, gRNA oligonucleotides were annealed to generate fragments containing the gRNA spacers flanked by BsaI recognition sites. These fragments were then digested with restriction enzymes and ligated to vectors containing a promoter and terminator for gRNA expression. The resulting ligation products were introduced into E. coli DH5 cells, which were plated and cultured to form colonies. The plasmids were extracted from the agar plates. Pooled gRNA plasmids were extracted from the colonies and introduced into yeast cells. PCR flanking the region of interest was performed to generate the amplicons representative of the yeast gRNA libraries. Nanopore sequencing of the amplicons were performed using SQK-RBK110.96.