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1.Description ..........................................................................................................22.Product Components and Storage Conditions ............................................23.pGL3 Vector Maps and Sequence Reference Points . (2)
A.3B.pGL3-Enhancer
<4C.5D.64.Cloning Methods .. (7)
A.7
B.Preparation of pGL3 Vectors and Insert DNA 8
C.Transformation Protocols for 8
D.
Isolation of Plasmid DNA (8)
5.Transfection of Mammalian Cells ..................................................................96.Assay of Luciferase Activity ............................................................................97.Sequencing of Luciferase Reporter Vectors .. (11)
8.Appendix ...........................................................................................................12A.Common Structural Elements of the pGL3 Luciferase
Reporter Vectors ................................................................................................12B.Advantages of the 13C.The pGL3 Vectors luc + 14D.Mapping Genetic Elements Located Within 16E.Composition of Buffers 17G.pGL3-Basic Vector .18H.pGL3-Enhancer Vector 20I.pGL3-Promoter Vector 23J.pGL3-Control Vector 26K.Related Products. (28)
pGL3 Luciferase Reporter Vectors
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1.Description
The pGL3 Luciferase Reporter Vectors(a–c)provide a basis for the quantitative
analysis of factors that potentially regulate mammalian gene expression. These
factors may be cis-acting, such as promoters and enhancers, or trans-acting,
such as various DNA-binding factors. The backbone of the pGL3 Luciferase
Reporter Vectors is designed for increased expression, and contains a modified
coding region for firefly (Photinus pyralis) luciferase that has been optimized for
monitoring transcriptional activity in transfected eukaryotic cells. The assay of
this genetic reporter is rapid, sensitive and quantitative. In addition, these
Luciferase Reporter Vectors contain numerous features aiding in the structural
characterization of the putative regulatory sequences under investigation.
2.Product Components and Storage Conditions
Product Size Cat.# pGL3-Control Vector20μg E1741 pGL3-Basic Vector20μg E1751 pGL3-Promoter Vector20μg E1761 pGL3-Enhancer Vector20μg E1771 Information on related products, including the Luciferase Assay System, is provided in
Sections 4–7 and 8.K.
Storage Conditions:Store the pGL3 Luciferase Reporter Vectors at –20°C.
3.pGL3 Vector Maps and Sequence Reference Points
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素描技法
The listings of restriction sites for the pGL3 Luciferase Reporter Vectors are
provided in Section VIII.G–J.
Note: The specific transcriptional characteristics of the pGL3 Vectors will
vary for different cell types. This may be particularly true for COS cells,
which contain the SV40 large T antigen. The SV40 large T antigen promotes
replication from the SV40 origin, which is found in the promoter of the
pGL3-Promoter and pGL3-Control Vectors. The combination of large T antigen
and SV40 origin will result in a higher copy number of these vectors in COS
造船技术cells, which in turn may result in increased expression of the reporter gene compared to other cell and vector combinations.
Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA
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3.A. pGL3-Basic Vector
The pGL3-Basic Vector lacks eukaryotic promoter and enhancer sequences,allowing maximum flexibility in cloning putative regulatory sequences.
Expression of luciferase activity in cells transfected with this plasmid depends on insertion and proper orientation of a functional promoter upstream from luc +. Potential enhancer elements can also be inserted upstream of the promoter or in the BamHI or SalI sites downstream of the luc + gene.
cradle 2 the grave
Figure 1. pGL3-Basic Vector circle map. Additional description: luc +, cDNA
encoding the modified firefly luciferase; Amp r , gene conferring ampicillin resistance in E. coli ; f1 ori, origin of replication derived from filamentous phage; ori, origin of replication in E. coli.Arrows within luc + and the Amp r gene indicate the direction of transcription; the arrow in the f1 ori indicates the direction of ssDNA strand synthesis.
pGL3-Basic Vector Sequence Reference Points:Promoter (none)Enhancer
(none)Multiple cloning region 1–58Luciferase gene (luc +)88–1740GLprimer2 binding site 89–111SV40 late poly(A) signal 1772–1993RVprimer4 binding site
592aa2080–2061
ColE1-derived plasmid replication origin 2318β-lactamase gene (Amp r )3080–3940f1 origin
4072–4527upstream poly(A) signal 4658–4811RVprimer3 binding site
4760–4779
Promega Corporation ·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA
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3.B. pGL3-Enhancer Vector
The pGL3-Enhancer Vector contains an SV40 enhancer located downstream of luc + and the poly(A) signal. This aids in the verification of functional promoter elements because the presence of an enhancer will often result in transcription of luc + at higher levels.
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Figure 2. The pGL3-Enhancer Vector circle map. Additional description: luc +,cDNA encoding the modified firefly luciferase; Amp r , gene conferring ampicillin resistance in E. coli ; f1 ori, origin of replication derived from filamentous phage; ori,origin of plasmid replication in E. coli . Arrows within l
uc + and the Amp r gene indicate the direction of transcription; the arrow in f1 ori indicates the direction of ssDNA strand synthesis.
pGL3-Enhancer Vector Sequence Reference Points:Promoter
(none)Multiple cloning region 1–58Luciferase gene (luc +)88–1740GLprimer2 binding site 89–111SV40 late poly(A) signal 1772–1993Enhancer
2013–2249RVprimer4 binding site
2307–2326
ColE1-derived plasmid replication origin 2564β-lactamase gene (Amp r )3329–4186f1 origin
4318–4773upstream poly(A) signal 4904–5057RVprimer3 binding site
5006–5025
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