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: [log in to unmask]
*******************************************
----- Original Message -----
From: "Cynthia Kinsland" <[log in to unmask]>
To: "Jacob Keller" <[log in to unmask]>
Cc: <[log in to unmask]>
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: [log in to unmask]
>> *******************************************
>
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