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. 2022 Apr 6;14(4):806. doi: 10.3390/pharmaceutics14040806

Figure 1.

Figure 1

Graphical overview of the generation of the rPA83m expression vector. pQE-30-rPA1+2 and pQE-30-rPA3+4 are expression vectors containing nucleotide sequences of rPA1+2 and rPA3+4, respectively, that were cloned between the BamHI and HindIII cleavage sites. The rPA1+2-coding sequence is represented by blue rounds and the rPA3+4-coding sequence is represented by green rounds. The rPA1+2 3′-end sequence included in the rPA3+4Fwd primer is represented by the oval. Primers are indicated with the colour of the matrix to which they are specific. SphI and PstI restriction sites are located within the rPA1+2 and rPA3+4 nucleotide sequences, respectively. Restriction sites used for one-step cloning are marked in red. PCR1 is a product corresponding to the 3′-fragment of rPA1+2. PCR2 is a product corresponding to the 5′-fragment of rPA3+4 and also contains a 20-nucleotide length sequence that is specific to the 3′-end of rPA1+2. PCR3 is a fusion product generated by overlap PCR using PCR1 and PCR2 as primary fragments and SphIFwd and PstRev as flanking primers. 1% agarose gel electrophoresis (ethidium bromide staining) of purified PCR1 and PCR2, as well as a PCR mix containing amplified PCR3, is presented; the lengths of DNA ladder (GeneRuler DNA Ladder Mix, Thermo Scientific, Waltham, MA, USA) are indicated on the right.