expression vectors

Brian Osborne bosborne at nature.Berkeley.EDU
Sun Apr 17 21:22:20 EST 1994


Previously I wrote :


> To the Group,
> I am looking for an inducible expression vector for S.
> cerevisiae. It could be either 2 micron or CEN, preferably
> with some sort of polylinker for cloning of the cDNA, and
> without its own ATG. The vectors I used many years ago in
> L. Guarente's lab are clumsy and uncharacterized. I'm
> going to express some N. tabacum and Arabidopsis cDNAs
> in yeast, mostly exploratory experiments. I apologize
> if this question is asked too often.
> Please respond to me and I will gladly summarize.
>
> Thank you for your attention to this matter,
>
> Brian Osborne
> bosborne at nature.berkeley.edu

And I received the following responses, many thanks to :

mj at sequencer.wustl.edu (Mark Johnston)
"Dave Eide"  <deide at ua.d.umn.edu>
Peter Pavlik <PP at abc.univie.ac.at>
"David Gonda" <David_Gonda at quickmail.cis.yale.edu>

==================================================

Hi Brian:

        I can send you a "kit" of plasmids containing the GAL1-10
promoter.
 I have a plasmid (pBM150 or 272) where the GAL promoter provides the
RNA start site, and the inserted gene provides the ATG, and plasmids
(pBM756, 758) where the GAL fragment provides both the RNA start site
and the ATG codon.  The cloning sites are RI and HindIII.  I have
distributed these plasmids widely, so you may be able to obtain them
from Jasper Rine or Jeremy Thorner (talk to Jeff Flick in Thorner's
lab).  ]

Sincerely,

        Mark Johnston

==================================================

I have a recommendation for your vector request.  pRS316-GAL1 described
in Genetics 132 665-673.  We have used the yeast cDNA library
constructed in this vector with great success.

Dave

==================================================

Check pYES2 vector from INVITROGEN corp. It is URA3, 2 micron, Amp,
ColE1, f1 ori, GAL1 promotor, T7 promotor, polylinker, CYC1 terminator.

Good luck                                  Peter

==================================================

>We have both CEN and 2 micron vectors which have the EcoRI-BamHI
GAL10/1 >promoter fragment cloned into a pUC19 MCS, either type with
URA3, TRP1, or LEU2 >(these were made from the Geitz and Sugino set of
vectors; YCplac22, YCplac33, >YCplac111, YEplac112, YEplac181,
YEplac195). The unique sites available >downstream from the GAL1
promoter are BamHI, XbaI, SalI, PstI, SphI, and >HindIII. Also, the
still unique EcoRI site is just downstream from the GAL10 >promoter
(GAL1 and GAL10 promoters are essentially the same, but divergently
>transcribed). Both promoters are off in glucose, strongly induced by
galactose. >We intend to construct analogous constructs using a
methionine regulatable >promoter, but don't have those yet. If you would
like our YCplacGAL and >YEplacGAL constructs or have additional
questions please email to below. > >regards, > >David Gonda > >




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