title: Design 3_Q5 with 5 cycle at Ecoli stage_SR3
design: First, oligo pool consists of single stranded gRNA, BsaI restriction sites and common handle was constructed. A short oligo complementary the oligo pool was used to extend and synthesize the second strand. The PCR cycle and DNA polymerase were varied. 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.