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The resulting 4.3 kb pHimar3 plasmid (containing the HimarFT transposon, the transposase gene, the neomycin phosphotransferase gene under the control of Francisella groEL promoter and the E. coli-compatible origin of replication oriR6K) was transformed into E.coli DH5αλ pir, and selection of transformants was performed on LB agar containing 50 µg ml−1 of kanamycin.

Stabilization of yeast transformants was performed with passaging on selective and non-selective media according to Klabunde et al. (2003).

The cold stress tolerance assay of E. coli transformants was performed using the method of a previous study (Hu et al. 2016).

Culture of transformants was performed in liquid selective medium (lacking uracil).

Genomic PCR on independent transformants was performed, and 15 independent lines of T0 tobacco transformants were obtained.

Selection of primary transformants was performed on the basis of resistance to hygromycin (30 µg ml−1).

Colony PCR and back-transformation into E. coli using plasmid preparations from putative transformants were performed to confirm the presence of recombinant plasmids in the yeast.

Experiments with stable transformants were performed with T3 generation IR α-dox plants.

To validate the observation that both fusion tags appear to increase accumulation levels of the recombinant protein in transgenic plants, statistical analyses of the primary transformants were performed.

Electrotransformations of JWCB005 and JWCB018 with unmethylated pDCW89 from E. coli or isolated plasmid DNA from C. hydrothermalis transformants were performed as described [ 14].

Lipofection transfection of E. histolytica trophozoites with the constructed plasmids, drug selection, maintenance of selected transformants, and indirect immunofluorescence analysis of independent established transformants were performed as described previously (5).