pBCN41-R4R3
(Plasmid #34916)

• Purpose: (Empty Backbone)
• Depositing Lab: Ben Lehner
• Publication: Semple et al Nat Methods. 2012 Jan 30;9(2):118-9. doi: 10.1038/nmeth.1864.

Backbone

• Vector backbone: pBCN21-R4R3
• 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::EGFP::myo-2_3'UTR transgene in the backbone that expresses well in various Caenorhabditis species.
• Vector type: Worm Expression
• Selectable markers: Neomycin (select with G418), Puromycin

Growth in Bacteria

• Bacterial Resistance(s): Chloramphenicol and Ampicillin, 25 & 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): ccdB Survival
• 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.
• Copy number: Unknown

Resource Information

• Supplemental Documents:
  • GenBank file
• A portion of this plasmid was derived from a plasmid made by: 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). EGFP gene from pPD129.57 (Addgene).

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
• Industry Terms:
  • Not Available to Industry

Depositor Comments

Dual resistance (PuroR-NeoR) vector for drug selection following biolistic bombardment in worms.
This vector contains a Pmyo-2::EGFP::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 pBCN40-R4R3 which has a red 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).