Under what circumstances would asymmetric cloning be desirable, with the inset having a different restriction site at each end?

Early gene-cloning experiments involved insertion at one restriction site in the vector; for example, the insert would have an EcoRI site at each end, and the vector would be opened at an RI site prior to ligation.

Choose all that apply.

1. Cloning in a fixed direction prevents the vector from becoming ligated to itself and forming either a circle or an end-to-end aggregate.

2. The orientation in one direction might be free because there is a desire to express a cloned gene driven from a promoter located near either one or another end of the link between vector and insert. The 3' end of the sense strand would need to be located downstream from the promoter.

3. The orientation in one direction should be fixed because there is a desire to express a cloned gene driven from a promoter located near one end of the link between vector and insert. The 5' end of the sense strand would need to be located downstream from the promoter.

4. Cloning in a fixed direction promotes the vector to become ligated to itself and form a double stranded ladder-type aggregate.