So, I would like to tag the Whi5 protein of the Saccharomices Cerevisiae. I would like to do it by PCR: first I want to multiply the GFP-kanMX casette part of the pym27 plasmid with primers that have homolog arms with the sequence of the Whi5. After this I want to use this PCR product for transformation thus tagging the Whi5 protein in the yeast cells.
I think I should design the forward primer in a way that it is not directly before the GFP because of the spatial arrangement/orientation, so both the Whi5 protein and the GFP can form the proper stucture.
Unfortunately, there is not one answer to this. There are many types of linkers you can use with each with their own purpose and function. A good place to start is: Article Fusion Protein Linkers: Property, Design and Functionality
There would be no proper answer, it depends on the protein, but the best is to use a construction that has been described in the literature. For example, i found this article
Article Whi5 Regulation by Site Specific CDK-Phosphorylation in Sacc...
that uses a GFP tagged whi5, you may search for others and use that construction, so you can be sure that it will work- Badges
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