A plasmid that carries two antibiotic resistance genes, to ampicillin

and tetracycline, respectively, has one restriction site for EcoRI located within the tetracycline resistance gene. Eukaryotic DNA containing a gene of interest (g.o.i) has been digested with different restriction enzymes, none of which cut within the gene of interest sequence). The resultant fragments are added to plasmid DNA that has been digested with EcoRI only; the mixture was then incubated with DNA ligase. Resulting plasmids (recombinant and otherwise) are added to competent bacteria lacking resistance genes and growing in the nutrient broth…
3. In order for this plasmid to incorporate the g.o.i. the eukaryotic DNA must have-
a. has been subjected to the action of several restriction enzymes b. been digested with EcoRI before mixing with the plasmid
c. been cut by any restriction enzyme that cuts above and below the g.o.i.
d. a tetracycline resistance gene included.

and if you could explain that would help a lot

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