Hi Jacob,
If you're including the TEV site in your primer, then you won't need a
restriction site after it (unless you're planning to recycle the
vector afterwards for other inserts).
Having the protease site too close to the protein can often make it
difficult to cut.
Cheers,
Charlie
Quotfintg Jacob Keller <[email protected]>:
I checked out the Sheffield et al paper, and the restriction sites
there are all just after the TEV site, thereby including, as
Cynthia
mentioned, at least an extra H beyond the obligatory G from the TEV
site. I was hoping to be able to have only the G. (Since I am
cloning
in the TEV site with my PCR primer, I have free choice about what
codons to choose, and therefore think it would be nice to have the
restriction site in the TEV site itself, if possible. Also, this
will
keep my primer a little shorter.)
Jacob
*******************************************
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
Dallos Laboratory
F. Searle 1-240
2240 Campus Drive
Evanston IL 60208
lab: 847.491.2438
cel: 773.608.9185
email: [email protected]
*******************************************
----- Original Message ----- From: "Cynthia Kinsland"
<[email protected]>
To: "Jacob Keller" <[email protected]>
Cc: <[email protected]>
Sent: Friday, June 05, 2009 5:19 PM
Subject: Re: [ccp4bb] TEV nucleotude sequence with restriction site
I'm not quite sure what you want, but I have a series of vectors
encoding various N-terminal tags and fusions, all followed by a
TEV
site. They have an MCS standard to many pET vectors.
Therefore,
they are designed to clone your gene in using the NdeI site at
the
5' end (which will, after proteolysis, leave you with GH at the
N-terminus of your protein). Other restriction enzymes in the MCS
can be used, but more amino acids will be left at your
N-terminus.
I've used WatCut (from the U. Waterloo) for the silent
mutagenesis
question: http://watcut.uwaterloo.ca/watcut/watcut/template.php
Best,
Cynthia
On Jun 5, 2009, at 5:41 PM, Jacob Keller wrote:
Dear Crystallographers,
Does anybody have a TEV-protease-site-coding nucleotide sequence
with a commonly-used restriction site in it, preferably right at
the end? Alternatively, does some somebody know of a program to
determine all equivalent codon permutations for a small coding
region, filtered for resulting restriction site possibilities?
It
seems like it would be an easy enough script to write...
(I have already done some googling around for such a program,
with
not much luck.)
Jacob
*******************************************
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
Dallos Laboratory
F. Searle 1-240
2240 Campus Drive
Evanston IL 60208
lab: 847.491.2438
cel: 773.608.9185
email: [email protected]
*******************************************
--
Charlie Bond
Professorial Fellow
University of Western Australia
School of Biomedical, Biomolecular and Chemical Sciences
M310, 35 Stirling Highway
Crawley WA 6009, Australia
