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: j-kell...@northwestern.edu
*******************************************
----- Original Message -----
From: "Cynthia Kinsland" <cl...@cornell.edu>
To: "Jacob Keller" <j-kell...@md.northwestern.edu>
Cc: <CCP4BB@JISCMAIL.AC.UK>
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: j-kell...@northwestern.edu
*******************************************
