Addgene - pLVPT-GDNF-rtTR-KRAB-2SM2 Plasmid Data

Gene/insert name:		mPGK, GDNF, rtTR-KRAB-2SM2, Tet-Off
Alternative names:		GDNF
Insert size (bp):		Unknown
Gene/insert aliases:		GDNF, ATF1, ATF2, HFB1-GDNF
Species of gene(s):		H. sapiens (human)
Vector backbone:		pLVPT-rtTR-KRAB-2SM2 (Search Vector Database)
Type of vector:		Mammalian expression,Lentiviral
Backbone size (bp):		11516
Cloning site 5':		See map
Site destroyed during cloning:		No
Cloning site 3':		See map
Site destroyed during cloning:		No
5' Sequencing primer:		See map (List of Sequencing Primers)
Bacteria resistance:		Ampicillin
High or low copy:		High Copy
Grow in standard E. coli @ 37C:		No
Please specify bacterial strain for growth and growth condition:		Use Stbl3 or HB101 to reduce chance of recombination. Grow at 37C
Sequence:		View sequence
Author's Map:		View map
Plasmid Provided In:		Stbl3
Principal Investigator:		Patrick Aebischer
Terms and Licenses:		MTA, Tet Systems LULL

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 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.

Addgene has sequenced a portion of this plasmid for verification. Click here for the sequencing result.

Click on map to enlarge

Selected features

HIV-1_5_LTR

455 - 635

truncHIV-1_3_LTR

455 - 635

HIV-1_psi_pack

746 - 790

RRE

1300 - 1533

ORF frame 1

1639 - 2301

cPPT

2032 - 2047

MSCV_rev_primer

2242 - 2219

mPGK_F_primer

2616 - 2635

ORF frame 2

2723 - 3358

pBluescriptSK_primer

3381 - 3397

IRES

3421 - 3924

ORF frame 3

3987 - 4994

pBluescriptKS_primer

5080 - 5064

WPRE

5102 - 5689

cPPT

5760 - 5775

U3PPT

5760 - 5781

TRE

5812 - 6099

HIV-1_5_LTR

6131 - 6311

truncHIV-1_3_LTR

6131 - 6311

Sp6_primer

6354 - 6337

pBRrevBam_primer

6663 - 6644

tet(564-300)

6855 - 6592

pGEX_3_primer

6996 - 7018

AmpR_promoter

7177 - 7205

ORF frame 2

7247 - 8107

Ampicillin

7247 - 8107

pBR322_origin

8262 - 8881

pBABE_3_primer

9127 - 9107

SV40_enhancer

9328 - 9113

SV40_promoter

9125 - 9393

SV40_origin

9292 - 9369

SV40_promoter

9245 - 9447

SV40pro_F_primer

9354 - 9373

SV40_int

10574 - 10589

SV40_3_splice

10595 - 10642

SV40_PA_terminator

11218 - 11349

EBV_rev_primer

11306 - 11325

Unique restriction sites

NotI		1145
ClaI		2147
BclI		2896
SmaI		3401
BstBI		5039
SacII		5603
MscI		5702
XhoI		5812
FspI		7811

Article: A versatile tool for conditional gene expression and knockdown. Szulc J et al. (Nat Methods. 2006 Feb . 3(2):109-16. Pubmed)

Please acknowledge the principal investigator and cite this article if you use this plasmid in a publication.

Also, please include the text "Addgene plasmid 11647" in your Materials and Methods section. This information allows Addgene to create a link from the plasmid page to your publication.