Stratagene's pBK-CMV?

Marianna Max drmax at casbah.acns.nwu.edu
Thu Mar 31 20:22:27 EST 1994


In article <2na50n$qah at mserv1.dl.ac.uk>, bates <dbates at hph.ucdavis.edu> wrote:
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>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
>
I'm using the RSV version of this vector. They say that for eukaryotic
expression it is best to remove the prokaryotic promoter. I cut with Nhe1
and if I remeber correctly, with Spe in the polylinker and religated the
compatible ends to make an eukaryotic only expression vector. This remove
about 200bp and leaves most of the polylinker while removing any ambigous
start sites.

I haven't transformed my cells yet so I can't tell you how well it works.

Max





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