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Detection of binding sites is performed mapping back the sequenced fragments to the genome (Fields, 2007).
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The 5′ upstream region of Os04g27430 in RH, KD, and IL162 was sequenced (accession nos. KC511031, KC527594, KC511035), and searches for transcription factor (TF) binding sites were performed (http://www.cbrc.jp/research/db/TFSEARCH.html).
Prediction of kinase binding sites was performed with http://scansite.mit.edu/motifscan_seq.phtml.edu/motifscan_seq.phtml
Prediction of the putative transcriptional factor binding sites was performed using Transfac software.
Analysis of transcription factor binding sites was performed using GENOMATIX software (http://www.genomatix.de).de
Confirmation of the predicted binding sites was performed using Quantitative real-time PCR (Q-PCR).
Search for conserved transcription factor binding sites was performed by combining the PromAn, TFSearch and MatInspector programs [39], [40].
An analysis of individual TFBS with a test for pair wise co-occurrences of binding sites was performed.
Site-directed mutagenesis of the individual transcription factor binding sites was performed and the mutated sequences were cloned upstream of luciferase reporter gene in pGL3 Basic plasmid.
Inspection of this sequence for the presence of consensus transcription factor binding sites was performed by high stringency searches using PATCH (Pattern search for transcription factor binding sites) and TRANSFAC 6.0.
A genome wide scan of CAP for H-NS binding sites was performed by a pattern search, using the high affinity site of E. coli K12 [52], with a maximum of two mismatches.
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