Hello Jacob,
If no extra residue other than G or S is desired, I think the answer is LIC.
Or you may have to put the whole TEV site plus the restriction site on the
5' primer.
Here is the TEV site that I designed for our vector (The left half is
probably the only possible LIC sequence for ENLYFQG, so no options for
codons. But it does work in human cells):
ggg gaa aat tta tat tta aat aat ata taa ccc
The ATTTAAAT in the middle is a SwaI site.
When doing the LIC, prepare the vector by cutting it with SwaI.
On your cloning PCR primers, you need to have sequences like these:
Example: 5'with Glycine only(minimum):
aa aat tta tat ttc cag ggc NNN NNN NNN NNN NNN NNN
Example: 3'with TAA (minimum)
ttatatattatttcc tta NNN NNN NNN NNN NNN NNN
After PCR, put your cut vector and PCR product in a PCR tube, add 1x klenow
buffer, 50uM dGTP, 1~5U klenow fragment, treat at RT for 15min, then
deactivate at 65C for 10min, then slowly cool the mixture to anneal the
ends. Then transform E coli.
But also, let me mention this: restriction enzymes BspEI and BamHI only give
you G-S or S-G, which probably would not be worse than a G only. As I
remember, TEV works even better if the site is ENLYFQ-S.
Zhijie
----- Original Message -----
From: "Jacob Keller" <j-kell...@md.northwestern.edu>
To: <CCP4BB@JISCMAIL.AC.UK>
Sent: Friday, June 05, 2009 6:29 PM
Subject: Re: [ccp4bb] TEV nucleotude sequence with restriction site
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
*******************************************
