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. 2021 Aug 20;11(16):e4133. doi: 10.21769/BioProtoc.4133

Figure 2. Features of the pET28a(+) plasmid and position of oligonucleotides used in the two PCR protocols.

Figure 2.

The pET28a plasmid includes the φ10 (T7) promoter and the lac operator, as well as the translation initiation region (TIR) encompassing the Shine-Dalgarno (SD) sequence, a spacer, and the first five codons of the open reading frame. Protocol 1 oligonucleotides (blue) incorporate four nucleotides within the T7 promoter at +3-6 relative to the mRNA transcriptional start site. Protocol 2 oligonucleotides (purple) incorporate nucleotide mutations in the TIR, which increases protein production titers.