I'm struggling to understand the Golden Gate scheme. I dont work with TAL effectors, I need to make a repetitive string of small fluorescent proteins.
If I understand correctly, you need the overhang part on each end of each of the pieces you want to connect? so how having those four letter overhangs doesnt mess up with my reading frame? and also, im getitng four random letter every repeat in my protein?
They also say the enzymes like BsaI leave no scar, but they have one base that needs to be constant, so how come there is no scar?
I would like to have something like this:
Could you, please, give me a for-dummies explanation on how to design primers?
I have the piece that is my actual gene to be repeated, [ATG-fluorescent_protein-TAA]
I think, if I understand it correctly, I need to get several sets of PCR primers and copy that gene with primers introducing recognition sites for Bsal (or similar restriction enzyme) and overhangs to connect my pieces in a special manner. But then, those overhangs I use stay there. How can a 4 letter overhang be used and not mess up reading frames for repeated genes?
Thank you very much for all help!