A high-copy E. coli plasmid that DOESN'T use the pMB1 origin

Use this category for questions directly related to DNA manipulation (isolation, purification, sequencing, etc.) and questions regarding general PCR methodologies

Moderators: r.rosati, mchlbrmn

A high-copy E. coli plasmid that DOESN'T use the pMB1 origin

Postby PaulHDear » Sep 24 2016 4:11 pm

Hi all.

Does anyone know a high-copy E. coli plasmid that doesn't use the pMB1 origin of replication? pBR322, pUC, pTYB, Litmus, pColE1 and pBluescript all seem to use the same origin, which includes some restriction sites for enzymes that I want to use for other purposes. Surely someone, somewhere must have found an alternative high-copy replication origin? My undying gratitude* to anyone who knows of one!

(This claim has not been evaluated by the FDA; your mileage may vary.)

Paul
PaulHDear
newcomer
newcomer
 
Posts: 1
Joined: Sep 24 2016 4:06 pm

Re: A high-copy E. coli plasmid that DOESN'T use the pMB1 or

Postby atet » Dec 19 2016 10:52 pm

R6Kgamma origin + pir116 mutat gene
atet
newcomer
newcomer
 
Posts: 1
Joined: Dec 19 2016 6:28 pm

Re: A high-copy E. coli plasmid that DOESN'T use the pMB1 or

Postby 29yrsExperience » Sep 14 2017 9:23 am

If you can tolerate a "medium" copy number plasmid, there is an old one called pACYC184 (Chang and Cohen, 1978, J. Bacteriol 134, 1141-1156) that has a p15A origin. it is ~4.2 kb. The authors describe it as having a copy number of about 20. It can be amplified with spectinomycin. It does not have a MCS, but you can clone into a few unique sites within its two drug resistance genes, for chloramphenicol or tetracycline. There's an EcoRI in the CmR gene, for instance. Maybe someone has made an improved vector based on this one. I have found it useful as it is compatible with the pMB1/colE1 replicons. I think it is still available commercially, I'm but not sure.
29yrsExperience
technophile
technophile
 
Posts: 34
Joined: Aug 11 2017 12:58 pm


Return to DNA and General PCR Methods

Who is online

Users browsing this forum: No registered users and 1 guest