pBI Tet四环素诱导双基因表达载体质粒 BioVector NTCC质粒载体菌种细胞基因保藏中心

pBI Tet四环素诱导双基因表达载体质粒 BioVector NTCC质粒载体菌种细胞基因保藏中心
The pBI Tet Vector is a response plasmid that can be used to express two genes of interest from one bidirectional tet-responsive promoter (Pbi-1; 1) in Clontech’s Tet-On® and Tet-Off® Gene Expression Systems and Cell Lines (2). The Tet Expression Systems and Cell Lines give researchers ready access to the tetracycline-regulated expression systems described by Gossen & Bujard (3; Tet-Off) and Gossen et al. (4; Tet-On). The pBI Tet Vector contains the bidirectional promoter Pbi-1 which is responsive to the tTA and rtTA regulatory proteins in the Tet-Off and Tet-On systems, respectively. Pbi-1 contains the Tet-responsive element (TRE), which consists of seven copies of the 42-bp tet operator sequence (tetO). The TRE element is between two minimal CMV promoters (PminCMV), which lack the enhancer that is part of the complete CMV promoter. Consequently, Pbi-1 is silent in the absence of binding of TetR or rTetR to the tetO sequences. PminCMV-1 and PminCMV-2 control the expression of two separate genes of interest. Note that the cloned inserts must have an initiation codon. In some cases, addition of a Kozak consensus ribosome binding site (5) may improve expression levels; however, many cDNAs have been efficiently expressed in Tet systems without the addition of a Kozak sequence.
pBI allows the simultaneous regulation of two genes of interest by one central TRE. After a stable
Tet-On or Tet-Off cell line has been established by transfecting with a tTA or rtTA regulator plasmid,
pBI is cotransfected with pTK-Hyg (#6153-1) to permit selection of a double-stable cell line which
expresses both genes of interest. Alternatively, pPUR (#6156-1) or another selection plasmid can
be used. If this plasmid contains an enhancer element, as does pPUR, cointegration of pBI and the selection plasmid may lead to higher background expression. Double-stable, tet-responsive cell lines with pBI
response constructs can be developed using the protocols described for pTRE response plasmids in the Tet Systems User Manual.

Location of Features
• Multiple cloning site (MCS II): 4345–6
• Pbi-1 Bidirectional Tet-responsive promoter: 12–568
PminCMV-2: 122–12
Tet-responsive element (TRE): 128–439
PminCMV-1: 440–568
• Multiple cloning site (MCS I): 603–637
• Fragment containing the β-Globin poly A signal: 644–1811
• Col E1 origin of replication: 2012–2655
• Ampicillin resistance gene
β-lactamase coding sequences: 3663–2803
• Fragment containing the SV40 poly A signal: 4328–3877
Propagation in E. coli
• Suitable host strains: DH5α and other general purpose strains.
• Selectable marker: plasmid confers resistance to ampicillin (50 µg/ml) on E. coli hosts.
• E. coli replication origin: Col E1

References
1. Baron, U., et al. (1995) Nucleic Acids Res. 17:3605–3606.
2. Tet Expression Systems and Cell Lines (July 1996) CLONTECHniques XI(3):2–5.
3. Gossen, M. & Bujard, H. (1992) Proc. Natl. Acad. Sci. USA 89:5547–5551.
4. Gossen, M., et al. (1995) Science 268:1766–1769.
5. Kozak, M. (1987) Nucleic Acids Res. 15:8125–8148.

【Supplier来源】BioVector NTCC Inc.
TEL：+86-010-53513060
【Website网址】 http://www.biovector.net