I wanted to clone eGFP into the plasmid next to IRES and PUROr gene but noticed CMV promotor is way far from the cloning site, does it work, or do I need to add promote as well when I include the GFP region.
please suggest
Dear Krishnaraju,
You can add GFP following an additional IRES (or T2A) sequence. Although adding two or more IRES sequences might decrease the efficacy of ribosomal skipping, but it can still work. In this case, T2A sequences have been shown to work better.
CMV is a very good promoter. It can be even better if you add any regulatory sites(enhancers I see, regulatory regions, regulation of the ORF) but the promoter should not be far from the gene you want to express. You can still do, and it will work, but if is too far away, you may not get the expression needed. It is always best to aim to be closer to the promoter and above all the regulatory regions. You can insert the GFP in the MCS and aim for AmpR but maybe you don't get in those genes. (What I can't see if you mean in between. Do you think the GFP can be inserted in PfIMI 2356?) maybe you could take the whole insertion into the MCS, and maybe treat each one separately. I see the gag protein. It could work even more efficiently if It is closer,and I know it is truncated. (Is there a special interest on the GFP? Maybe by its selection.?Maybe not for this kind of plasmid or protein? )You have MMLV and Lac promoter. MMLV gives benefits for the plasmid. Lac promoter is a high efficient promoter (should you consider having more elements for the plasmid that are from Lac?)
Thank you. Please come and gives us feedback. We would like to know what you think.
Hi Alireza and Angeles, thank you for the reply. my concern is why the promoter is far away from the MCS and MMLV and gag has something to do with it.
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