Pyramiding of the modifications/constructs created by the PCR-fusion/Gateway procedure. An example for pyramiding of gene modifications engineered by the PCR-fusion/Gateway procedure Showing the construction of an AtCesA7 cDNA mutant in which all eight Cys codons are mutated to Ser. Note that the pyramiding scheme described could be applied for all described cloning protocols. (A) General Cys residue structure of the Zn-finger domain of the CESA proteins. The two pairs of Cys residues which are mutated separately are designated. (B) Schematic of pyramiding of the Cys-residue mutagenesis in the Zn-finger domain of AtCesA7. Step 1: two AtCesA7Cys>SercDNA mutants are constructed in parallel, starting from wt AtCesA7 cDNA using the 'site-directed mutagenesis' protocol (Fig. 4a). In mutant expression clone 1 the codons of first pair of Cys are converted to Ser and in clone 2 the last pair. The mutated cDNA from expression clones 1 and 2 are generated using BP clonase and Gateway donor vector (pDONR/Zeo) to obtain mutant entry clones 1 and 2. Terminal regions from each of the mutated entry clones are PCR amplified with specific primers containing mutated codons of the remaining internal Cys residues of the Zn-finger. Overlap extension and cloning of the assembled DNA fragment into p3KC binary destination vector resulted in expression clone 3 in which all eight Cys codons are mutated to Ser. (C) Sequence analysis of the region of the Zn-finger domain mutant in expression clone 3. The converted Cys>Ser residues are marked with (*) and replaced nucleotides are shown in brackets.