This other DNA molecule is often used in the form of a vector, which could be a plasmid, a bacteriophage, a derived cosmid or phagemid, a transposon or even a virus. Techniques should also be available, which will allow selection of chimeric genomes obtained after insertion of foreign DNA from a mixture of chimeric and the original vector (see later).

Another critical desired feature of any cloning vector is that it should possess a site at which foreign DNA can be inserted without disrupting any essential function. Therefore, in each case an enzyme will also have to be selected which will cause a single break.

Sometimes vectors are modified by inserting a DNA segment to create unique site(s) for one or more enzymes to facilitate its use in gene cloning. This inserted DNA with restriction sites for several enzymes is sometimes called a polylinker.