Neomycin resistance gene in E. coli stb3 cells We use a lentivirus packaging vector called pLenti_puro_DEST. We use puromycin to select for the cloned gene in 293T cells. However, the pLenti_Neo_Dest has been discontinued, so I'm cloning a Neomycin resistance gene into the pLenti_Puro_Dest thus replacing Puromycin with Neomycin. I usually just grow up this gene in Stb3 or Stb4 cells from invitrogen. I'm wondering if I can select for cloned pDEST vectors in these cells using Neomycin or G418. According to the G418 manual, you can theoretically use it for bacterial selection, but I think that may be for libraries and not actually growing up the plasmid. So is it possible to select for my plasmid in these cells by adding a little G418 to the ampicillin plate? Is this common? I can't find anyone selecting bacteria by Neomycin anywhere. EDIT - The main reason I ask, is that the resistance gene is under a PGK promoter that I have no idea if it works in bacterial cells.

PGK is mammalian (murine) phosphoglycerate kinase promoter. It will therefore not work in bacterial systems. Why don't you select using ampicillin only? You can increase the ampicillin concentration if you want.

Regarding:

> I can't find anyone selecting bacteria by Neomycin anywhere.

Kanamycin is generally used for bacterial selection. The Neomycin resistance gene works on Neomycin, Kanamycin and G418 (a gentamycin variant). The gene basically codes for an aminoglycoside phosphotransferase and different variants of the gene have different affinities for different aminoglycoside substrates. For a basic reference see this.