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" <[log in to unmask]>
To: <[log in to unmask]>
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: [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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