Genes conferring neomycin-resistance are commonly included in DNA plasmids used to establish stable mammalian cell lines expressing cloned proteins in culture. Many commercially available protein expression plasmids contain a neo-resistance gene as a selectable marker. Biosynthetic pathway
Neomycin was first isolated from the Streptomyces fradiae and Streptomyces albogriseus in 1949 (NBRC 12773). Neomycin is a mixture of neomycin B (framycetin); and its epimer neomycin C, the latter component accounting for some 5–15% of the mixture. It is a basic compound that is most active with an alkaline reaction. It is also thermostable and soluble in water (while insoluble in organic solvents). Neomycin has good activity against gram-positive and gram-negative bacteria, but is ototoxic. Its use is thus restricted to the oral treatment of intestinal infections.Neomycin B is composed of four linked moieties: D-neosamine, 2-deoxystreptamine (2-DOS), D-ribose, and L-neosamine. Neomycin A, also called neamine, contains D-neosamine and 2-deoxystreptamine. Six genes are responsible for neamine biosynthesis: DOIS gene (btrC, neo7); L-glutamine:DOI aminotransferase gene (btrS, neo6); a putative glycosyltransferase gene (btrM, neo8); a putative aminotransferase (similar to glutamate-1-semialdehyde 2,1-aminomutase) gene (btrB, neo18); a putative alcohol dehydrogenase gene (btrE, neo5); and another putative dehydrogenase (similar to chorine dehydrogenase and related flavoproteins) gene (btrQ, neo11).
A deacetylase acting to remove the acetyl group on N-acetylglucosamine moieties of aminoglycoside intermediates (Neo16) remains to be clarified (sequence similar to BtrD).Next is the attachment of the D-ribose via ribosylation of neamine, using 5-phosphoribosyl-1-diphosphate (PRPP) as the ribosyl donor (BtrL, BtrP); glycosyltransferase (potential homologues RibF, LivF, Parf) gene (Neo15).Neosamine B (L-neosamine B) is most likely biosynthesized in the same manner as the neosamine C (D-niosamine) in neamine biosynthesis, but with an additional epimerization step required to account for the presence of the epimeric neosamine B in neomycin B.

Neomycin C can undergo enzymatic synthesis from ribostamycin. Composition
Standard grade neomycin is composed of several related compounds including neomycin A (neamine), neomycin B (framycetin), neomycin C, and a few minor compounds found in much lower quantities. Neomycin B is the most active component in neomycin followed by neomycin C and neomycin A. Neomycin A is an inactive degradation product of the C and B isomers. The quantities of these components in neomycin vary from lot-to-lot depending on the manufacturer and manufacturing process. DNA binding
Aminoglycosides such as neomycin are known for their ability to bind to duplex RNA with high affinity. The association constant for neomycin with A-site RNA is in the 109 M−1 range. However, more than 50 years after its discovery, its DNA-binding properties were still unknown. Neomycin has been shown to induce thermal stabilization of triplex DNA, while having little or almost no effect on the B-DNA duplex stabilization.