Figure 4.

Approach to mutate a predicted cis-element: Generation of a construct in which a predicted cis-element (shown in red) is changed (shown in blue) involves two rounds of PCR. In the first PCR the target gene with two primers sets (F1/R1 and F2/R2). The F1 and R1 primer set amplifies the gene from the initiation codon to the predicted cis-element and F2 and R2 will amplify from the predicted cis-element to the stop codon. Primers F1 and R2, in addition to gene-specific sequence (shown in green), will be tailed with sequences complementary to Gateway vector primers (shown in dark yellow). Similarly, primers R1 and F2, in addition to gene-specific sequence, will be tailed with the changed sequence in the predicted cis-element (shown in blue). In the second PCR, the two gene fragments from the first PCR will be mixed. This overlapping template will be amplified using primers complementary to primers F1 and R2 tailed with the attB1 and attB2 Gateway sequences, which can then be cloned into a Gateway donor vector and into a plant transformation vector with a tag as a fusion to the N-terminus. The wild type gene will be cloned in a similar fashion except that only one PCR will be done with the F1/R2 primer set containing the entire attB1 and attB2 Gateway sequences.