pSLGCV
(Plasmid #49862)

• Purpose: Expression in Caenorhabditis elegans of the luc+ gene (Promega), fused in frame to GFP (S65C), under the sur-5 promoter (from plasmid pTG96, John Yochem and Min Han). Backbone pPD95.79 (Firelab).
• Depositing Labs: Anne Glover, Jonathan Pettitt
• Publication: Lagido et al BMC Physiol. 2008 Apr 2;8:7. doi: 10.1186/1472-6793-8-7.

Backbone

• Vector backbone: pPD95.79
• Backbone manufacturer: Andrew Fire, Carnegie Institution for Science
• Modifications to backbone: added sequentially the sur-5 promoter [36], PCR-amplified from plasmid pTG96 and then the luciferase gene PCR-amplified from vector pSP-luc+ (Promega). A 3.7 kb fragment upstream of the sur-5 gene containing the promoter, was amplified using primers 5'- ATAAGCTTGCATGCCTGCATTGC-3' and 5'-AGACACCCCGGGCTTTCTGAAAAC-3' (this introduces a Sma I site and removes the start codon for the sur-5 gene). The sur-5 promoter was inserted in the pPD95.79 backbone using appropriate restriction sites (Sph I and Sma I). The luc+ gene was amplified using primers 5'-TCCCGGGAAGCTTTCCATGGAAGAC-3' and 5'-CTAGGGTACCACGGCGATCTTTCC-3'. Sma I and Kpn I were used to insert luc+ downstream of the sur-5 promoter and upstream of and in frame with gfp. The peroxisome tagging sequence is absent from luc+ and the NLS for sur-5 was excluded from pSLGCV, so that LUC+::GFP was expressed in the cytoplasm
• Vector type: Worm Expression

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: firefly luciferase luc+ fused to GFP (S65C)
• Species: Photinus pyralis
• Promoter: sur-5
• Tag / Fusion Protein:
  • GFP (S65C) (C terminal on backbone)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: SmaI (unknown if destroyed)
• 3′ cloning site: KpnI (unknown if destroyed)
• 5′ sequencing primer: 5'-TCCCGGGAAGCTTTCCATGGAAGAC-3'
• 3′ sequencing primer: 5'-CTAGGGTACCACGGCGATCTTTCC-3'

Resource Information

• A portion of this plasmid was derived from a plasmid made by: vector backbone pPD95.79 from Firelab, Carnegie Institution for Science. pTG96 plasmid (from which promoter was PCR- amplified) obtained from John Yochem and Min Han; University of Colorado, Boulder. vector pSP-luc+ (bought from Promega. Luc+ gene was PCR-amplified from here.
• Articles Citing this Plasmid:
  • 3 References

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
  • Luciferase Limited Use Label License
• Industry Terms:
  • Not Available to Industry

Depositor Comments

Although no formal MTAs signed with Firelab or University of Colorado Boulder, we accepted that the material was provided for academic research only.