I am making a construct for a plasmid that transfect HEK293T. The thing I want to include in it is CD9 transmembrane protein, target peptide, and 6xHistag.I don't understand where 6xhistag is added? Linker sequence will be added to the fused target peptide with CD9 or not. How two proteins are fused? If a linker is used, which one linker will be added? Plz guide how to make construct for plasmid for genetic engineering of exosome
Are you using snap gene or GENEtyx for this design?
It's much easier to make a construct with this software!
If histag has both N-terminus or C-terminus transport signals then you can add histidine to the coding sequence like this:
pCMV- Histag-CD9-GFP/RFP
or pCMV-CD9-Histag-GFP/RFP
Once the construct is done, get your primers, proceed to PCR and purify plasmid for transient expression.
After confirming that CD9 is expressed on the plasma membrane then you can now transfer the insert into a stable expression vector. Hopefully, your 293T cells should then overexpress CD9 compared to endogenous CD9 at 17-22kDa and you should be able to detect histag on the same band as CD9.
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