Re: [DIYbio] Design a plasmid

It depends on your type of plasmid replication, but for "Rolling Circle
Mechanism" (RCM), which most cloning vectors use, you need an origin
sequence and a protein called "rep". So, your rep protein will be coded
for by a promoter, rbs and coding sequence, with a terminator. In most
cloning vectors, these are compressed into a big block called "ori", but
analyse them and you'll find an open reading frame coding for a rep protein.

In many cloning vectors, this is all you'll find, but with RCM plasmids
you also should have a "single stranded origin" (SSO), which
unfortunately goes by many other unrelated names. This is a sequence
that folds into a promoter-like shape when the DNA is single-stranded,
and triggers RNA priming of single-stranded DNA. This RNA primer is then
used to copy the complementary strand of the plasmid.

Without this SSO sequence, you'll end up with loads of single-stranded
plasmids as the DNA is copied from the main origin, but without an SSO
they don't efficiently get converted to double-stranded DNA again. This
is one of the reasons why many commercial or "traditional" cloning
vectors are so unstable without antibiotic selection all the time.

On 03/02/13 12:34, Mega wrote:
> Hi,
>
> Independently of the plasmid for the glowing plants, I'd like to design
> an *entirely* new plasmid just for fun/curiosity.
>
> Therefore a few questions:
>
>
> The replication origin, does it need to have a promoter? I assume not so?
>
> The backbone does it have to have a special length? or as short as
> possible (synthesis costs, ease of transformation, etc.)
>
>
> Best,
> Andreas
>
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