Hi all,
I would like to clone one gene with a fluorescent tag into a vector containing NatMX marker. In my current lab, we only have pFA6-NatNT2 plasmid. http://www.euroscarf.de/plasmid_details.php?accno=P30346
Plasmid map shows that it contains "ori", so it would be maintained as a plasmid in the cell. However, I would like to integrate it to the genome. Would it be enough just removing the "ori" region from the plasmid to make it integrative? Or should I order a new plasmid instead?
Cheers, Arman
That's probably bacterial ori. The plasmid should work fine in my opinion.
Hi Tadej Markus ,
Thank you for your answer. For sure plasmid has to contain bacterial "ori" site for its replication in for example E.coli. But I am not 100% sure if it is for yeast or for bacteria. I guess I have to Blast the sequence.
That ori will undoubtedly be for E. coli, so if you want to use as an integrative vector in yeast then it probably will be fine. If you want to integrate it into E. coli, that is more difficult. You can't just delete the ori because then you have no way to actually prepare your plasmid for any manipulations.
Dear Michael J. Benedik ,
Thank you for your answer. I have found that the "ori" sequence in this plasmid is for bacterial replication as you said.
Cheers,
Arman
Successively replace the ori in the plasmid with a heat-sensitive one. This will allow selection of integrands. If you are using E. coli
to increase the efficiency, you can use the lambda phage system.
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