SD sequence of di-cistronic cloning

[Rosalind324]

I wonna construct a di-cistronic cloning, pET-22b(+) vector, and insert gene II behind gene I (both need SD sequence and termination codon),

but I was so confused. How can I determine the place (the bp length)between the termination codon of gene I and SD sequence at the begining of gene II?no place(follow on the heels of gene I) or add some inessential base pairs?

Any suggestion?or some protocols recommended?

Thank in advice.

[LilKim]

ya know.. that's an interesting question... I've actually used vectors that are di-cistronic ... but I can't remember what if there was an "intervening sequence"

I guess i could suggest that maybe you should look at (non-fusion) comercial vector that had a coding region for GFP next to the MCS .. and see if/how long the interveneing sequence is. (remember to keep everything in-frame if you're going to add a sequence)

good luck!
- KIM

[Rosalind]

I appreciate it, KIM.

But I still have something intangibly.

I have consult some series of non-fusion vectors such as pBAT4 (GST, non-fusion) which has two promoter with directon conversely

In fact, I just want to construct two-gene expression cloning with one promoter, because I hope gene I protein ahead could help gene II protein soluble, just in the manner of co-expression.
and I have no idea how much interval between them.That 's the key of the question.

by the way, all thing I mentioned above is about prokaryote(E.coli).

Thank in advice.

[seaseasea]

hi,

at present,i am also constructing a vector which will have ten genes under one promoter.

the interveneing sequence could be either long or short(no interveneing sequence).
you can make two genes coupled,ie.,the stop codon of the first gene could be TAA,and the start codon of the second gene is ATG,so we can use TAATG as a stop codon and a start codon simultaneously by shareing a A base.in this case,there are no SD for the second gene.a advantage of this is the ratio of the first gene to the second gene is one to one.
another strategy is long interveneing sequence.
if the secong gene have its own SD,the distance of interveneing sequence is not important.5,10,15,20bp are all OK.in this case,bacause of synthesizing of two genes individually,distance is not important.

best wishes!

if you have other problem,please contact me at free.E-mail:lydiao@sibs.ac.cn