Re: [DIYbio] Transposon Design

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If you don't add a terminator, you could be activating or silencing
(by transcribing mRNA reverse compliments) all kinds of genes in the
locality of your inserted transposon; this could have huge knock-on
effects for the cell, and consequently for your desired DNA's behaviour.

So, always include a terminator. :)

A bi-directional terminator would be a bonus, too, to make sure your
DNA isn't read into by some external promoters on the + or - strand.

If you had space, you could even put your favourite bi-directional
terminator on both ends of your transposon casette to fully insulate
your genes from read-through to either end.

That way, if your only clone happens to have your transposon shoved in
the middle of an active operon, at least it'll stop the operon from
interfering with your cassette (even if the messed-up-operon may have
downstream effects that *do* mess things up..).

On 04/07/2013 09:39 AM, Mega wrote:
> Hi,
>
> I may get the chance to get to work with a transposon,
>
> however I have a design question:
>
>
>
> Do I need to include a terminator? Because the DNA is inserted
> anywhere in the E.Coli genome, and somewhere afterwards there is a
> terminator for sure.
>
> For the basical design I'd ligate three PCR products together, and
> the terminator would be the fourth fragment to attach -> can I save
> the working time? :D
>
>
> Would you just go on without?
>
>
> Best,
>
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