I am trying to design a CRISPR KO line for my gene of interest but the problem is that removing my protein of interest seems to kill the cells. I need a vector that has a Tet-On promoter, so that I can induce the Cas9 and gRNA whenever I want and another constitutive promoter that can express the selection marker. I would like to select after the transfection before inducing the KO.
hi, why dont u try pTRIPZ vector. this vector have TRE promoter and also UBC (constitutive promoter)
I recommend you to use pLIX_403 vector, which is Tet-on Gateway lentiviral vector with Puromycin selectable marker but the promoter is hPGK (not CMV).
I don't think it is practicable, because CAS9 cDNA is very large and TET elements also large.They exceed the capacity of lentivirus
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