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Merging gene (protein N-terminus with 6xHis) and clone to vector?


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#1 Dancia

Dancia

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Posted 08 November 2014 - 05:42 AM

Hello. I need to clone a gene into a vector. Gene must be merged with His tag at protein N-terminus.
I am trying to clone with program SnapGene.
 
This is my first time even trying to clone something into a vector and I have a few problems.
1. How do I know where actually is my N-therminus in a gene. I know I can translate gene, but the protein sequence itself gives me 0 information where is C or N end.
This is my gene with translated protein http://s24.postimg.o...klegl/image.png (the green box indicates where I believe I should cleave)
2. Now for example I believe my N-therminus is near the green box (above link). I should cleave somewhere at this point and then
in my vector near his: http://s24.postimg.o...ltsvp/image.png I could insert this insert like this: http://s24.postimg.o...yvf5h/image.png near
 his tag. The problem now would be the terminal stop conod, which would not let me translate his tags.
 So this approach in general is bad?
 
 My second idea is to use my own primer with 6xHis and ignore those 6xHis which are on  my vector. Then I would end with:
promotor> 6xHis-gene-stop
 
Any suggestion and ideas how to solve this problem?

Edit: I did some research.
I believe I can cleave at NspI (first picture - my gene) and add CAT-CAT-CAC-CAC-CAC-CAT. I would end with market N-terminus gene. But now how do I insert it into vector? + It would leave sticky ends?

Edited by Dancia, 08 November 2014 - 06:02 AM.





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