Fig. 1.

Vector engineering schema illustrating the creation of the plasmid pWGHisAmp from pTSHQn (Promega), and the NESG modified pET15 vector. PCR and In-Fushion^TM ligation independent cloning methods were used to replace the ORF and HQ metal affinity tag in pTSHQn with the pET15 linker region and 6xHis metal affinity tag to generate pWGHisKan. This was subsequently followed by replacement of the kanamycin resistance gene in pWGHisKan with the pET15 ampicillin resistance gene to generate the final vector pWGHisAmp. PCR derived products can now be cloned into either the NESG modified pET15 bacterial expression plasmid or into the pWGHisAmp plasmid for wheat germ cell-free expression