pSEAP2-Basic載體描述 pSEAP2-Basic allows for expression of the reporter gene secreted alkaline phosphatase (SEAP). This vector lacks eukaryotic promoter and enhancer sequences and has an MCS that allows putative promoter DNA fragments to be inserted upstream of the SEAP gene. Enhancers can be cloned into either the MCS or unique downstream sites. The SEAP coding sequence is followed by the SV40 late polyadenylation signal to ensure proper, efficient processing of the SEAP transcript in eukaryotic cells. A synthetic transcription blocker (TB), composed of adjacent polyadenylation and transcription pause sites, located upstream of the MCS reduces background transcription (1). The vector backbone also contains an f1 origin for single-stranded DNA production, a pUC origin of replication, and an ampicillin resistance gene for propagation and selection in E. coli. The SEAP2 Vectors incorporate a number of features that improve the sensitivity of SEAP by increaing the efficiency of SEAP expression or that enhance the utility of the vectors. These include: an improved Kozak consensus translation initiation site (2); the removal of the SV40 small-t intron, which can cause cryptic splicing and reduced expression in some genes and/or cell types (3, 4); switching from the early to late polyadenylation signal of SV40, which typically causes a five-fold increase in mRNA levels (5); an expanded multiple cloning site (MCS); compact plasmid size; and removal of extraneous sequences from the 3' untranslated region of the SEAP mRNA.
The integrated set of Great EscAPe? SEAP2 Vectors has been designed to provide maximal flexibility in studying regulatory sequences from the gene of interest. pSEAP2-Basic allows for expression of SEAP under control of putative promoters and/or enhancers of interest. The secreted SEAP enzyme is assayed directly from the culture medium and permits time-course studies not possible with assays dependent on cell lysates. Furthermore, the cells can be used for further investigations such as RNA or protein studies. The SEAP2 Vectors can be transfected into mammalian cells by any standard method.
Propagation in E. coli
Suitable host strains: DH5α and other general purpose strains. Single-stranded DNA production requires a host containing an F' episome such as JM109.
Selectable marker: plasmid confers resistance to ampicillin (100 μg/ml) to E. coli hosts.
E. coli replication origin: pUC
Copy number: ~500
Plasmid incompatibility group: pMB1/Col E1 References: 1. Eggermont, J. & Proudfoot, N. (1993) EMBO J. 12:2539–2548.
2. Kozak, M. (1987) Nucleic Acids Res. 15:8125–8148.
3. Huang, M. T. F. & Gorman, C. M. (1990) Mol. Cell. Biol. 10:1805–1810.
4. Evans, M. J. & Scarpulla, R. C. (1989) Gene 84:135–142.
5. Carswell, S. & Alwine, J. C. (1989) Mol. Cell. Biol. 9:4248–4258.
6. Levitt, N. et al. (1989) Genes Dev. 3:1019–1025.
7. Enriquez-Harris, P. et al. (1991) EMBO J. 10:1833–1842.
Note: The attached sequence file has been compiled from information in the sequence databases, published literature, and other sources, together with partial sequences
obtained by Clontech. This vector has not been completely sequenced