I am planning on cloning a construct expressing shRNAs into a commercial pCMV-LifeAct-TagGFP2 plasmid. However, potential restriction sites for cloning new elements into this construct are very limited.
One of the few potential unique restriction sites (NdeI) is in the middle of the CMV enhancer element preceding the actual CMV promoter. Do you think it would be possible to interrupt the CMV enhancer element by cloning a fragment of 300 bp into this site, and still get expression from the CMV promoter? The CMV promoter is really strong in the cells I am using, so a small decrease in the transcription levels can be tolerated, but a complete loss of expression would ruin the experiment.
Any help will be highly appreciated!
You can find the sequence of the LifeAct-GFP plasmid here:
For automated annotation, you can use PlasMapper (note that CMV Enhancer (1-508) is not recognized by PlasMapper):
Submit your paper to J Biol Methods today!
Is CMV enhancer essential for protein expression from CMV promoter?
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