Hello everyone,
I am looking for a plasmid which has the MCS separate from the reporter, I would like to express my protein and GFP (but any fluorescence protein would be ok) togheter in the same cells but not as a fusion protein. I looked in addgene but I could not find something like this (but maybe there is and I just cannot find it). Any advice? If you can suggest plasmids you have worked with directly it would be better!
Thanks!
PS: I would like a plasmid for mammalian expression, which can be transfected with chemical methods, preferentially gateway (but not necessary).
Any vector containing an IRES will by fine for your scope. One promoter and cap dependent and IRES depentent translation for reporter and target. Here is a list of some
http://www.clontech.com/IT/Products/Fluorescent_Proteins_and_Reporters/Coexpression/IRES_Bicistronic?sitex=10023:22372:US
pGL3 "basic" Luciferase Vector is a promoter-less vector (used to characterize of regulatory sequences) you can add another promoter (you can use SV40 promoter and a CMV promoter) downstream of the 3'UTR and poly-A signal of your protein.
You could try either:
1) A construct with an MCS-2A-GFP cassette under a single promoter, since the 2A self-cleavage peptide will allow for the final proteins to not be fused together.
2) A bicistronic construct with the MCS under one promoter, and the reporter gene under a separate promoter entirely. As long as both promoters are constitutive and known to be validated in your cell type, both proteins should be expressed.
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