Question

To ensure that your cloned s gene is inserted into the expression vector in the correct orientation, you use 'double enzyme cloning' (i.e. adding different restriction enzymes sites to the 5' and 3' ends of the gene). given the available restriction enzyme sites in the multiple cloning site in the vector below, which combination of restriction enzymes will result in the s gene always inserting in the correct orientation relative to the promoter?

Answer 1

To ensure the cloned s gene inserts in the correct orientation, use two restriction enzymes like EcoRI and SalI at the 5' and 3' ends. This method allows the gene to be ligated into the vector in only one orientation.

Step-by-step explanation:

To ensure that your cloned s gene is inserted into the expression vector in the correct orientation, you must use two different restriction enzymes that recognize different sequences, one at each end of the s gene. Let's assume you have the following multiple cloning site in your vector:

• EcoRI site (5' end)
• BamHI site
• SalI site (3' end)

By designing your s gene with an EcoRI site at the 5' end and a SalI site at the 3' end, you can clone the s gene in such a way that it can only be inserted in one orientation - the correct orientation relative to the promoter. If you were to use the same restriction enzyme on both ends, the gene could be inserted backward. With different enzymes, the gene can only ligate into the vector with the promoter in the right direction, ensuring proper expression of the s gene. It's important to check that there are no internal sites for these enzymes in your gene, though, as this could result in unwanted cuts.