Using CRISPR/Cas9 and homoly directed repair I have knocked in a degron tag onto the start of a protein of interest in 293T cells. WB has confirmed that ~5% of protein alleles have recieved the KI. To increase expression, we wanted to use loxp/cre to remove the blasticidin casette after selection.

We infected with Cre and now the degron tagged protein is no longer being expressed at all. Once we are able to return to work (due to virus) we will do geneomic sequencing to figure out what is happening.

In the mean time I was looking for advice, possibly someone could look at my plamsid and see somethign wrong I am missing.