I'm currently designing a mammalian expression vector.
When I look up the sequence for my gene of interest on Genbank I notice that the sequence has 18 base pairs upstream from its Start (ATG) codon, and 180 base pairs after its Stop Codon (TGA).
DNA20 provides a really neat service to design a vector. I copy and pasted my Gene from its start codon to stop codon, and added the EcoR1 restriction sites to either side of this sequence.
Is this a common technique in the designing of a vector, or should I be including the full sequence of my gene from Genbank (base pairs before the start codon and after the stop codon)? I'm fairly certain that these regions wont be translated, but is it common practice to insert the whole sequence anyway?
Also, the vector I am using has a multiple cloning site with ten or so sites to choose from. None of those restriction sites exist in my gene of interest. I chose Eco R1 because we have plenty of this enzyme in our lab. But are there any restriction sites to avoid using in mammalian expression vector design.
any help on this is much appreciated