pLV-EGFP-Cre
(Plasmid #86805)

• Purpose: Lentiviral vector that expresses an EGFP-Cre fusion protein with a nuclear localization signal from the CMV promoter
• Depositing Lab: Amy Lasek
• Publication: Truitt et al eNeuro. 2016 Oct 31;3(5). pii: ENEURO.0256-16.2016. eCollection 2016 Sep-Oct.

Backbone

• Vector backbone: LV-Lac
• Backbone manufacturer: Inder Verma
• Vector type: Mammalian Expression, Lentiviral, Cre/Lox

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 30°C
• Growth Strain(s): NEB Stable
• Copy number: Unknown

Gene/Insert

• Gene/Insert name: Cre recombinase
• Species: bacteriophage P1
• Insert Size (bp): 1800
• GenBank ID: YP_006472.1
• Promoter: CMV
• Tags / Fusion Proteins:
  • EGFP (N terminal on insert)
  • nuclear localization signal: PKKKRKV (N terminal on insert)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: AfeI (destroyed during cloning)
• 3′ cloning site: SmaI (destroyed during cloning)
• 5′ sequencing primer: CMV

Resource Information

• Articles Citing this Plasmid:
  • 4 References

Terms and Licenses

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

Depositor Comments

The plasmid pEGFP-cre (~5.75 Kb) was a gift from Brigitte Kieffer and Gregory Scherrer and was generated while they were at the University of Strasbourg and the Institut de Génétique et Biologie Moleculaire et Cellular (IGBMC), Illkirch, France.

A nuclear localization signal (NLS) PKKKRKV was added after the ATG start site of EGFP-cre with QuikChange II XL Site Directed Mutagenesis kit and protocol (Stratagene # 200521), and confirmed by sequencing. The ~1.8 Kb AfeI/SmaI blunt NLS-EGFP-cre fragment was then removed and ligated into blunted BamHI sites of the ~6.7 Kb backbone from the lentiviral plasmid vector LV-Lac (Addgene #12108) ie LV-Lac with the Lac removed. This ligation was transformed into SURE2 cells. DNA from potential subclones was screened for orientation with restriction digestions and checked with sequencing. Unfortunately the sequencing showed that this cloning strategy created a false ATG/start site 6 AAs downstream of the true ATG start of the EGFP-cre. To eliminate any potential problems this false ATG start site was mutated to ATC with QuikChange II XL Site Directed Mutagenesis and confirmed by sequencing. The corrected pLV-EGFP-cre subclone was subsequently transformed into TOP10F’ which gave better DNA yields than those from SURE2.

This plasmid was produced as part of the NIAAA-funded INIA Consortium.

Additional reference (PMID: 27986929):
Harris et al (2016) Genetic and pharmacologic manipulation of TLR4 has minimal impact on ethanol consumption in rodents. J. Neurosci., 37(5):1139-115.