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All promoter variants were cloned upstream of the uidA coding region (GUS) to create promoter- GUS fusions.
When applied to a set of promoter, transcribed and intergenic sequences, they create promoter-rich and promoter-poor clusters.
To assist in the process of defining unique NF-Y complexes, we have created promoter:beta-glucuronidase fusion lines for all 36 Arabidopsis genes.
This consists of the combinatorial ligation of previously created promoter regions, i.e. sequences that correspond to the distal region (upstream the −35 box), to the core region (between the −35 and −10 boxes) and to the proximal region (downstream the −10 box).
Using this system, we created promoters with strengths ranging from 2% to 319% compared with ermEp1.
For measuring the promoter activities of operons within the maltose regulon upon their induction in single carbon limited- and mixed substrates cultures, we first created promoter-GFP reporter plasmid-containing E. coli cells, as shown in Additional file 2. The inducers, cAMP and maltotriose activate the promoters of the maltose regulon genes to synthesize GFP.
For introduction of a first RMCE-competent "donor cassette", the target unit contained the CMV promoter 5′ upstream of F3 to create a promoter trap in the following transfections.
To create a promoter swap construct in pGS156, the promoter region of GRE3 was amplified from CBS7001, fused by PCR to the GRE3 coding and the 3′ region from GSY4318 using a primer that contained overlapping sequence from the 3′ end of the promoters and the 5′ end of the open reading frame (GSP533), and cloned into pGS35.
In order to initiate functional characterization of CIP2A promoter, the −1802 bp to +182 bp 5' upstream region of CIP2A gene that was analyzed for SNPs above, was cloned into pGL4.10 vector to create CIP2A promoter luciferase reporter construct (−1802CIP2ALuc).
