I plan to use PCR to amplify a promoter(~800bp) from Arabidopsis genomic DNA
and then construct it into pCAMBIA1303 replacing the CaMV35S promoter. I
want to know should I amplify the promoter upstream the ATG start codon or
can I amplify it upstream the transcription initiation site? The 5'UTR is 80
bp long for my gene. We already have a construct with promoter containing no
5'UTR region and so the ATG codon within NcoI site is immediatley downstream
the promoter. If this construct is transformed into Arabidopsis, can it be
correctly transcribed and downstream reporter gene translated?
Any idea on promoter amplification is apprecaited!
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well it seems to me, that you just have to make sure, that the reading
frame is correct for both the gene-of-interest and the reporter genes.
In other words, your can virtually translate your construct from the
initiationcodon right untill the end of the reportergenes. (Both GUS
and GFP). Than check if this corresponds to the expected proteins. It
should be transcibed and translated as a fusionprotein. It will be a
very big one, I don't know if that will work properly.
Rob van den Bekerom
University of Utrecht