Dear Amira

If the GFP is fused at the C_term of your construct you can simply perform mutagenesis at the end term to add a sto codon or delection at the N-term to remove the GFP sequence.

Generelly i'm doinf it using the PIPE cloning approach.. by performing a vector PCR by using primers that containing overlapping regions and ampify the region of the vector that we would like to mantain but not the region that you would like to delete (eg GFP)

In the followng link you can find an example of primer desing to delete/replace signal peptides with this approach

https://proteocool.blogspot.com/2021/09/tips-of-week12-rapid.html

but the same could be done for deletion of the GFP

best regards

Manuele

I would recommend against this approach. Adding a stop codon at the end of your protein of interest that is tagged with GFP will likely create a nonsense mediated decay substrate since the cells will perceive that stop codon as a premature stop codon hence greatly reducing the expression of GOI. I would suggest designing primers that have a stop codon added to the primer tail and amplify your gene from the tagged vector and reclone into a fresh empty vector that does not contain the GFP sequences at all.

I agree with a hybrid of the answers already provided. Either design primers to amplify the desired GOI and clone into fresh backbone, or design primers to perform an inverse PCR to remove the c-terminal GFP and then kinase and ligate the amplicon to yield a "clean" GOI only construct.

Thank you very much for all replies, it is really appreciated. I have designed the primes including the stop codon directly after the sequence of my gene of interest and will apply mutagenesis to stop the expansion of eGFP.