Hello everyone; I am new to CRISPR.
Owning to our strategy to screen positive cells, we yearn to involve two selection markers (GFP and puromycin) in the LentiCRISPRv2 vector. However, we figure out that the viral titer is low in this way, and the possible explanation is the large size of the vector. Also, we noticed that there are several non-functional sequences (?) in this vector. The main question is: Is it ok to remove the highlighted sequence (including EM7 promoter, BleoR, SV40polyA signal, lac promoter, and CAP binding site) in the file?
We think this strategy might be practical since the selection strategy in prokaryotic system in based on Ampicillin, and Zeocin (BleoR) should be the a trademarker, which is actually non-functional?
Thanks a lot
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