Addgene - pLVPT-GDNF-tTR-KRAB Plasmid Data

Gene/insert name:		mPGK, GDNF, tTR-KRAB, Tet-on
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-tTR-KRAB (Search Vector Database)
Type of vector:		Mammalian expression,Lentiviral
Backbone size (bp):		11510
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

TetR

4002 - 4601

ORF frame 3

3987 - 4994

pBluescriptKS_primer

5080 - 5064

WPRE

5096 - 5683

cPPT

5754 - 5769

U3PPT

5754 - 5775

TRE

5806 - 6093

HIV-1_5_LTR

6125 - 6305

truncHIV-1_3_LTR

6125 - 6305

Sp6_primer

6348 - 6331

pBRrevBam_primer

6657 - 6638

tet(564-300)

6849 - 6586

pGEX_3_primer

6990 - 7012

AmpR_promoter

7171 - 7199

ORF frame 2

7241 - 8101

Ampicillin

7241 - 8101

pBR322_origin

8256 - 8875

pBABE_3_primer

9121 - 9101

SV40_enhancer

9322 - 9107

SV40_promoter

9119 - 9387

SV40_origin

9286 - 9363

SV40_promoter

9239 - 9441

SV40pro_F_primer

9348 - 9367

SV40_int

10568 - 10583

SV40_3_splice

10589 - 10636

SV40_PA_terminator

11212 - 11343

EBV_rev_primer

11300 - 11319

Unique restriction sites

NotI		1145
ClaI		2147
SmaI		3401
BstBI		5039
SacII		5597
MscI		5696
XhoI		5806
FspI		7805

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 11646" in your Materials and Methods section. This information allows Addgene to create a link from the plasmid page to your publication.