Stratagene's pBK-CMV?

bates dbates at hph.ucdavis.edu
Tue Mar 29 16:02:15 EST 1994



<<<<<< Attached TEXT file follows >>>>>>
Does anyone out there have any experience using Stratagene's pBK Phagemid
vectors? We are planning to use one of them (pBK-CMV) for eukaryotic expression
and it looks like a nice vector, except for one thing. It has an ATG for
expression of LacZ in bacterial cells, but this ATG is surrounded by what looks
like a pretty good Kozac consensus sequence. Since this site will also be
transcribed in eukaryotic cells we are worried that we may end up with
translation starting too far upstream, so that at best we get a fusion protein
rather than the native peptide, and at worst we get garbage (if it's not cloned
in in frame).

We can engineer an Nhe1 site into our insert and use that to excise the ATG but
it will involve more mesing around than we hoped. Is  it ok to use without
doing this? Has anybody successfully used this vector to get expression of the
peptide coded by the insert DNA as opposed to some sort of fusion protein etc?

Your comments would be greatly appreciated

Thanks

Dave




More information about the Methods mailing list