Sounds like a great project! Ok, a few quick thoughts.

Which end of your receptor are you putting the fusion protein on? Do you have a knockout line that you can you to check for fusion protein function? Just keep in mind that it is possible to inhibit protein localization or protein function by adding on eGFP.

For your cloning strategy, check to make sure your fusion is in-frame and that you have removed any stop codons or else you'll just get the first protein.

I like to use NEBs cutter tool to check for enzyme sites in my genes, very handy when planning a new cloning strategy.

For primers, I order from IDT, which also let's you check melt temperatures.

If you have a map of your plasmid that would help folks here brainstorm with you.

Hope this helps!

Dear Amy Klocko,

Thanks so much for your insights and suggestions—they’re incredibly helpful!

• I’m planning to fuse the eGFP at the C-terminal of the receptor, as I want to track the protein's expression/localization without disrupting its function.
• Currently, I don’t have a knockout line, but I’ll explore that option to validate the fusion protein's functionality. Thanks for pointing that out.
• Absolutely, I’ll double-check for any stop codons and confirm the fusion remains in-frame during cloning. I agree that’s a critical step to avoid premature translation termination.
• For primers, I’ve also used IDT before, and I’ll definitely check melt temperatures there. Their tools make it straightforward.
• I can share a plasmid map once I finalize it—great idea to have others review and brainstorm further.

Really appreciate your detailed input—it gives me a lot to work with as I move forward. Thanks again!

Muhammad