Plasmid 34915: pBCN40-R4R3

• Gene/insert name: None
• Vector backbone: pBCN21-R4R3
  (Search Vector Database)
• Vector type: Worm Expression
• Modifications to Backbone: Contains the dual antibiotic resistance operon Prpl-28::PuroR::rpl-16_outron::NeoR::let-858_3'UTR instead of the puromycin resistance gene. Spe I, Xcm I, Bgl II, Avr I and Sac I restriction sites introduced into backbone for linearisation before bombardment. 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. Contains a new version of Pmyo-2::mCherry::myo-2_3'UTR transgene in the backbone that expresses well in various Caenorhabditis species.
• Bacterial resistance(s) Ampicillin and Chloramphenicol
• Growth strain(s) ccdB Survival
• Growth temperature (℃): 37
• Growth instructions: 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.
• High or low copy: Unknown
• Selectable markers: Neomycin, Puromycin
• Person or lab that originally
  cloned the gene/insert: Puromycin resistance gene from pBabePuro (Addgene). rpl-28 promoter and let-858 3'UTR sequences from pPD129.57 vector (Addgene). Neomycin resistance gene from pEGFP-C1 (Clontech). mCherry gene from pCFJ104 (Addgene).
• Sequence: View sequences (4)
• Map: View map
• Principal Investigator: Ben Lehner
• Terms and Licenses: MTA

Comments: 

Dual resistance (PuroR-NeoR) vector for drug selection following biolistic bombardment in worms.
This vector contains a Pmyo-2::mCherry::myo-2_3'UTR pharyngeal marker on the backbone. Worm strains generated with vector can be crossed with strains generated with other visual markers such as in pBCN41-R4R3 which has a green fluorescent pharyngeal marker.
This is a Gateway 3-fragment compatible destination vector. It contains AttR4 and AttR3 sites (not AttR1 and AttR2 sites as identified automatically by the Addgene algorithm).

Article: Generating transgenic nematodes by bombardment and antibiotic selection. Semple et al (Nat Methods. 2012 Jan 30;9(2):118-9. doi: 10.1038/nmeth.1864. PubMed)