Design a plasmid

[Mega]

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

[Cathal Garvey]

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.

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[Andreas Sturm]

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.

Ah, great. 

That's why it is so long I guess... 

[Koeng]

I "designed" a new plasmid as well.. except I used the gibson assembly. It is very useful, you should check it out. Take a bunch off stuff you want and just put it together! (gotta have some dna with the stuff of course ( for a template))

[Koeng]

I was thinking about this, and is there anywhere I can find the sequence for the rep protein? I was thinking about PCRing it out and  connecting it with different promoters to make inducable plasmid counts. (also does it work in bacteria? :)   )

[Robert Sidney Cox III]

You can find it in the SC101 literature, assuming you are talking about RepA.

Most high-copy cloning vectors don't have it (they use RNA regulation).