I need help with this Molecular Biology Question Develop a c

I need help with this Molecular Biology Question:

Develop a cloning strategy that would enable you to clone at a minimum the coding region from this sequence into the vector pUC19, so that the coding region is in a clockwise orientation (google “pUC19 sequence”). There are several ways to accomplish this cloning objective; the only requirement is that it is theoretically possible and that you can show that the coding region is in a clockwise orientation.

Here are the examples that were given in class, but I can\'t really think of any \"stradegies\" or I\'m having a hard time comprehending it:

You may assume that you have a reasonably competent lab technician to carry out the experimental work, so you don’t need detailed instructions for each experiment, however they do need instructions as to what to look for at each step. For example:

It is sufficient to say digest the sequence with restriction enzyme BstEII, and run a gel. They know how to do that, but you would have to tell them what sizes of fragments to look for on the gel. (If that information is important to your strategy.)

You can say transform bacteria with this DNA, and they know how to do that, however you would need to tell them what selection to use and, if you are using a blue/white screening system what color of colony they should look for.

Solution

One of the best strategies to predict the orientation of the insert in the plasmid is by sequencing the plasmid using M13 primers, which will give an idea about the orientation of the insert.

The other method using restriction enzyme would be to using one restriction enzyme specific of the vector (a unique site close to the cloning site) and one restriction specific of the insert. You then may calculate the distance between the two cleavage sites according to insert orientation relative to the vector. This will give the orientation of the insert in the plasmid.

The last method would be using PCR to amplify a certain region of the insert. The fragment orientation involves one primer specific of the vector (upstream or downstream of the cloning site) and on primer within the cloning sequence. If the primer set is correctly oriented, PCR will give a unique band with a specific size according to the distance between primers. If not properly oriented, there will be no amplification.