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 <[log in to unmask]>:
> 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]
>>> *******************************************
>>
--
Charlie Bond
Professorial Fellow
University of Western Australia
School of Biomedical, Biomolecular and Chemical Sciences
M310, 35 Stirling Highway
Crawley WA 6009, Australia
|