insert in the promoter region - (May/18/2004 )
I am trying to analyze the localization of one plant gene by putting a HA tag-fusion protein under the control of the native promoter. However, during my cloning, I have to add several bp (a restriction enzyme recognization site) between -1 and the ATG star codon. Do you think it will affect the normal regulation of the promoter and make the following localization inaccurate?
Certainly you can insert some sequence between the promoter and ATG. It won't have much effect on transcription. If you look at some report vector, you will find usually there is the multiple cloning region around the promoter.
just before the ATG is the kozak sequence. This is part of the translation initiation site. It is a somewhat conserved sequence like gccgccaccATG. Distroying it completly might result in lower expression (translation efficiency).
An Nco1 site is sometimes usefull: ccATGG