title: Design 1_Golden Gate_BsmBI at Ecoli stage_SR1
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. Direct plasmid sequencing was performed using SQK-RBK110.96.