PurposeTet-regulated (Tet-on) lentiviral vector for transgene (CAG promoter) - OR - shRNA (H1 promoter when subcloned from pLVTHM (Addgene#12247)) - 2nd generation
- Backbone size w/o insert (bp) 12877
Vector typeMammalian Expression, Lentiviral
Growth in Bacteria
Growth instructionsUse Stbl3 or HB101 to reduce chance of recombination. Grow at 37C
Copy numberHigh Copy
Gene/Insert nameCAG promoters, GFP, tTR-KRAB, Tet-on
- Cloning method Restriction Enzyme
- 5′ sequencing primer See map (Common Sequencing Primers)
Terms and Licenses
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
pLVTHM and packaging plasmids 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 the 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 discussions on cloning strategies and protocols.
These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which the plasmids were described, and include Addgene in the Materials and Methods of your future publications.
For your Materials & Methods section:pLVCT-tTR-KRAB was a gift from Patrick Aebischer & Didier Trono (Addgene plasmid # 11643)
For your References section:A versatile tool for conditional gene expression and knockdown. Szulc J, Wiznerowicz M, Sauvain MO, Trono D, Aebischer P. Nat Methods. 2006 Feb . 3(2):109-16. 10.1038/nmeth846 PubMed 16432520
Map generated by Addgene from full sequence supplied by depositor.
Map uploaded by the depositor.