pLVET-tTR-KRAB
(Plasmid #11644)

• Purpose: Tet-regulated (Tet-on) lentiviral vector for transgene (hEF-1alpha promoter) - OR - shRNA (H1 promoter when subcloned from pLVTHM (Addgene#12247)) - 2nd generation
• Depositing Labs: Patrick Aebischer, Didier Trono
• Publication: Szulc et al Nat Methods. 2006 Feb . 3(2):109-16.

Backbone

• Vector backbone: None
• Backbone size w/o insert (bp): 12386
• Vector type: Mammalian Expression, Lentiviral

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): Stbl3
• Growth instructions: Use Stbl3 or HB101 to reduce chance of recombination. Grow at 37C
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: hEF-1alpha, GFP, tTR-KRAB, Tet-on

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ sequencing primer: See map

Resource Information

• Addgene Notes:
  • Addgene diagnostic digest
  • Addgene diagnostic digest 2
• Articles Citing this Plasmid:
  • 5 References

Terms and Licenses

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

Depositor Comments

Transgenes can be expressed from any RNA Pol II promoter as part of
bicistronic unit comprising the KRAB-based repressor; tetO sequences
are inserted into the vector LTR. Tet-on and Tet-off versions rely on
repressors that bind in the absence or the presence of doxycycline,
respectively. Addition of Pol III promoter-small hairpin RNA cassette
allows for drug-controllable RNA interference (Tet-on shRNA).

Cloning shRNA into pLVET, pLVCT, and pLVPT vectors: first clone
shRNA into pLVTHM downstream of the tetO-H1 region. Then cut pLVTHM
containing your shRNA with MscI-FspI and clone the insert containing
the 3'LTR to target plasmid opened with MscI-FspI. FspI cuts into
AmpR. This means for inverted clones AmpR will not be restored; after
selection you will be left with clones with the shRNA cassette and
functional AmpR.

pLVTHM and packaging plasmid for this system are also available at Addgene http://www.addgene.org/rnaitools Please note that the full sequence for this plasmid is approximated and not fully verified. Please visit Trono lab website http://tronolab.epfl.ch to see frequently asked
questions on cloning strategies and packaging.
You may also visit LentiWeb http://www.lentiweb.com for discussion on cloning strategies and protocols.