Hi Vyacheslav,

yes, you can add introns to expression constructs that are efficiently spliced out - a fully artificial one should be possible, though it is probably risky. There are a number of features within the intron that help it to become spliced efficiently. If you want to ensure you have efficient splicing, why not introduce an intron that already exists in your host species? (one that has been validated etc).

I've used this approach before for my work in plants. I had constructs synthesised that contain an intron within a reporter gene (preventing bacterial expression), and used a plant intron that is recognised efficiently by multiple intron-prediction programmes.  In my case I had the construct synthesised, so also removed any potentially cryptic splice sites as well.

Hope this helps, good luck.

Nick

Thank you, I have a bacterial plasmid with two sequences that I need to join after the plasmid is replicated. This means that splicing should be post transcriptional and the regions to remove are pieces of plasmid - resistances, regulation sites etc. The splicing is performed by adding the nuclear extract to RNA, most frequently yeast or mammalian, right? Then I should include the splicing regulation sites into my bacterial plasmid, that are matching the requirements of that particular extract - yeast or mammalian, is that correct? So I am looking for essintial parts that I should add to my plasmid to facilitate correct splicing.

I'm not really sure what your application is, it seems that either you are making this more complicated than it needs to be, or you have an uncommon application.

"This means that splicing should be post transcriptional and the regions to remove are pieces of plasmid - resistances, regulation sites etc."

It isn't typical to have to remove all the parts of the plasmid for transfection experiments. How does the entire plasmid become RNA, to become spliced and remove all these things?

Typically, you would construct and propagate a plasmid in E.coli that was designed to be used for expression in a eukaryotic host. E.g. Pol-II promoter, cDNA or gene, terminator.  You can include introns within cDNA (e.g. from other genes) to prevent leaky expression of the gene in E.coli. This is often useful for reporter genes, so you can be sure the expression is from your transfected/transformed eukaryotic host.

"The splicing is performed by adding the nuclear extract to RNA, most frequently yeast or mammalian, right?'

This sounds very complex, and fraught with problems (RNA degradation for a start!) although possible. I'm sure that the original experiments investigating intron activity etc were probably performed like this.... but you wouldn't do this unless you were trying to investigate some subtle feature of intron behaviour.

If you do just want to put in an intron into your cDNA, to prevent leaky bacterial expression or to enhance translation, then find a well characterised (and hopefully smallish) intron from your host, and insert it within your cDNA sequence. Take lots of time doing this, because you can very easily cause frame shifts. You will also need to very carefully check that the itnron is properly recognised by various software, and not produce unwanted splice products.

Good luck, hopefully this helps. Apologies if I have misunderstood your application.

Nick

I am sorry I should start with the whole experiment explanation. I have a library of genes for a monomer of dimeric protein, cloned into the bacterial plasmid vector. I would like to create the library of sequences for homodimeric proteins from this set. Firstly I perform the transcription of all plasmids by rolling circle mechanism and then I would like to splice these long RNAs into the final sequences containing the genes for monomer aligned after each other (this covalent dimer is well know and characterized) - yielding the library of sequences with joined monomers. This is the reason why I need to remove the plasmid parts by splicing mechanism between the genes