Spe I, Xcm I, Bgl II, Avr I and Sac I restriction sites introduced into backbone for linearisation, if necessary.
The ccdB gene was replaced with a version from pDONR221 (Invitrogen) that is compatible with commercially available ccdB Survival E. coli, no longer requiring DB3.1 cells.
Ampicillin and Chloramphenicol
As a Gateway destination vector the empty vector must be grown in ccdB resistant cells such as DB3.1 or ccdB Survival. Use both Ampicillin and Chloramphenicol selection to avoid loss of the Gateway cassette.
Once the Gateway cassette has been replaced with the gene of interest select with Ampicillin in standard E. coli strain such as Top10 or DH5alpha.
Puromycin resistance gene from pBabePuro (Addgene). rpl-28 promoter and let-858 3'UTR sequences from pPD129.57 vector (Addgene).
Please acknowledge the principal investigator and cite this article if you use
this plasmid in a publication. Also, please include the text "Addgene plasmid
34917" in your Materials and Methods section.