$(i)$ Origin of replication : Origin of replication is a specific sequence from where replication starts.

When any piece of $DNA$ is linked to this sequence can be made to replicate within the host cells.

– This sequence is also responsible for controlling the copy number of the linked $DNA$.

So if one wants to recover many copies of the target $DNA$, it should be, cloned in a vector whose origin support high copy number.

$(ii)$ Selectable marker : In vector, there is selectable marker in addition to origin of replication.

$\Rightarrow$ Which helps in identifying and eliminating non transformants and selectively permitting the growth of the transformants.

Normally, the genes encoding resistance to antibiotic such as ampicillin, chloramphenicol tetracycline or kanamycin etc. are considered useful selectable markers for E. coli.

$(iii)$ Cloning sites: In order to link the

alien $DNA$, the vector needs to have very few, preferably single recognition sites for the commonly used restriction enzymes.

Presence of more than one recognition sites within the vector will generate several fragments, which will complicate the gene cloning.

The ligation of alien $DNA$ is carried out at a restriction site present in one of the two antibiotic resistance genes.