pTRE-Tight-BI双向四环素诱导调控表达载体质粒 BioVector NTCC质粒载体菌种细胞基因保藏中心

pTRE-Tight-BI双向四环素诱导调控表达载体质粒

pTRE-Tight-BI is a bidirectional TRE-Tight plasmid that can be used to inducibly express two genes (a combination of a reporter with a gene of interest or two genes of interest) simultaneously in our Tet-On and Tet-Off Gene Expression Systems and Cell Lines (1, 2). The Tet Expression Systems and Cell Lines provide researchers ready access to the tetracycline-regulated expression systems described by Gossen & Bujard (3; Tet-Off) and Gossen et al.(4; Tet-On). pTRE-Tight-BI vector contains a modified Tet response element (TREmod), which consists of seven direct repeats of a 36 bp sequence that contains the 19 bp tet operator sequence (tetO) (5; pTREtight). The two mini CMV promoters, which lack the enhancer that is part of the complete CMV promoter, flank the TREmod. The multiple cloning sites (MCS) I and MCS II flank the BI-Tet-responsive Ptight promoters on either side. Both genes inserted into MCS I and MCS II will be responsive to the tTA and rtTA regulatory proteins in the Tet-Off and Tet-On systems, respectively. Note that the cloned insert must have an initiating ATG codon. In some cases, addition of a Kozak consensus sequence (6) may improve expression levels; however, many cDNAs have been efficiently expressed in Tet systems without the addition of a Kozak sequence. pTRE-Tight-BI should be cotransfected with the Linear Hygromycin Marker (Cat. No. 631625, not included) or Linear Puromycin Marker (Cat. No. 631626, not included) to permit selection of stable transfectants
pTRE-Tight-BI Vector Vector InformationClontech Laboratories, Inc. www.clontech.com Protocol No. PT3893-52 Version No. PR083611The pTRE-Tight-BI-Luc Control Vector, packaged with the pTRE-Tight-BI vector, contains an additional 1,649 bp encoding firefly luciferase inserted into MCS I. This vector can be used as a reporter of induction efficiency using standard luciferase detection reagents with the gene of interest cloned into MCS II. pTRE-Tight-BI was derived from pTRE, (originally described as pUHD10-3 (5)) and pTREtight. Location of features• Ptight Tet-responsive promoter: –70–322Tet response element (TREmod): 3–252Location of seven tetO 19-mers: 12–30; 48–66; 83–101; 119–137; 155–173; 190–208 & 226–244Fragment containing PminCMV-1: 258–317TATAA box-1: 280–286 Fragment containing PminCMV-2: 2856–2788TATAA box-2: 2825–2819• Multiple cloning site I (MCS I): 335–405• Multiple cloning site II (MCS II): 2782–2726 • Fragment containing SV40 polyA signal-1: 417–617• Fragment containing SV40 polyA signal-2: 2726–2533• Fragment containing Col E1 origin of replication: 780–1379• Ampicillin resistance gene (β-lactamase): 2536–1540Sequencing primer locationspTRE-Tight Sequencing Primer:Reverse primer (683–660): 5'–TAT TAC CGC CTT TGA GTG AGC TGA–3'Propagation in E. coli• Suitable host strains: DH5α™ and other general purpose strains. • Selectable marker: plasmid confers resistance to ampicillin (100 μg/ml) in E. coli hosts.• E. coli replication origin: ColE1

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