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. 2017 Aug 5;7(15):e2431. doi: 10.21769/BioProtoc.2431

Figure 2. Schematics of the pAnti-GeneX construct.

Figure 2.

A. An example of a primer design yielding a spacer to target gene X. The targeted DNA is in pink. This protospacer is a 33 bp sequence in the gene of interest and is preceded by an AA, yielding a functional protospacer-adjacent motif (PAM). The forward and reverse primers encode annealing sequences to the pWUR477 template, and 5’ overhangs which together form the desired spacer sequence. B. PCR amplification with the forward and reverse primers on the pWUR477 plasmid and self-ligation to construct the pAnti-GeneX plasmid. Grey rectangles represent repeat sequences, pink represents the new spacer, orange represents spacer 2 and red represents spacer 3, and black represents the plasmid backbone.