pRL-null

价格:800元

联系方式:I47-825O-882O

买家导航

载体基本信息

出品公司: Promega
载体名称: pRL-null
质粒类型: 海肾荧光素酶报告载体;哺乳动物载体
高拷贝/低拷贝: 高拷贝
克隆方法: 限制性内切酶,多克隆位点
启动子: CMV
载体大小: 3320 bp
5' 测序引物及序列: CMV Forward: CGCAAATGGGCGGTAGGCGTG
3' 测序引物及序列: --
载体标签: --
载体抗性: 氨苄青霉素
筛选标记: --
克隆菌株: TOP10等常规菌株
宿主细胞(系): 哺乳动物细胞
备注: --
产品目录号: E2271
稳定性: 瞬表达 或 稳表达
组成型/诱导型: 组成型
病毒/非病毒: 非病毒

载体质粒图谱和多克隆位点信息

pRL-null载体图谱



pRL-null 多克隆位点

pRL-null 载体特征

载体简介

The pRL-null Vector(a,b) (Figure 1) is intended for use in constructing a control
reporter vector that may be used in combination with any experimental reporter vector
to cotransfect mammalian cells. All of Promega’s pRL Reporter Vectors contain a
cDNA(b) (Rluc) encoding Renilla luciferase, which was originally cloned from the
marine organism Renilla reniformis (sea pansy; 1). As described below, the Renilla
luciferase cDNA contained within the pRL Vectors has been modified slightly to
provide greater utility.
The pRL-null Vector contains no enhancer or promoter elements. Rather, it contains
a multiple cloning region upstream of Rluc to allow for the cloning of any desired regulatory
element(s) to drive expression of Renilla luciferase. Renilla luciferase is a
36kDa monomeric protein that does not require post-translational modification for
activity (2). Therefore, like firefly luciferase, the enzyme may function as a genetic
reporter immediately following translation. For information about the use of this plasmid
in conjunction with a reporter vector containing the firefly luciferase gene, refer
to the Dual-Luciferase Reporter Assay System(c,d) Technical Manual (#TM040).
The pRL Vectors are isolated from a dam–/dcm– E. coli K host strain, allowing
digestion with restriction enzymes that are sensitive to dam and dcm methylation.
The GenBank/EMBL Accession Number for the pRL-null Vector is AF025844.

Features of the pRL-null Vector

A. Multiple Cloning Region
The pRL-null Vector contains a multiple cloning region positioned immediately
upstream of the chimeric intron and Renilla luciferase reporter gene (Figure 2).
To aid in devising cloning strategies, Table 1 summarizes the types of DNA ends
generated from restriction endonuclease digestion within the multiple cloning
region as well as the compatibility of those ends with the ends of DNA fragments
generated by heterologous restriction enzymes.

B. Chimeric Intron
Downstream of the multiple cloning region of the pRL-null Vector is a chimeric
intron comprised of the 5′-donor splice site from the first intron of the human
β-globin gene, and the branch and 3′-acceptor splice site from an intron preceding
an immunoglobulin gene heavy chain variable region (3). The sequences of
the donor and acceptor splice sites, along with the branchpoint site, have been
modified to match the consensus sequences for optimal splicing (4).
Transfection studies have demonstrated that the presence of an intron flanking a
cDNA insert frequently increases the level of gene expression (5–8). In the
pRL-null Vector the intron is positioned 5′ to Rluc to minimize the utilization of
cryptic 5′-donor splice sites that may reside within the reporter gene sequence (9).

C. T7 Promoter
A T7 promoter is located downstream of the chimeric intron and immediately
precedes the Rluc reporter gene. This T7 promoter can be used to synthesize
RNA transcripts in vitro using T7 RNA Polymerase (Cat.# P2075). T7 RNA
Polymerase can also be used to synthesize active Renilla luciferase in a cell-free
coupled eukaryotic in vitro transcription/translation reaction (e.g., Promega’s
TNT Reticulocyte Lysate(c,e,f,g) [Cat.# L4610], TNT T7 Coupled Wheat Germ
Extract(c,e,f,g) [Cat.# L4140] or TNT T7 Quick Coupled Transcription/Translation
(c,e,f,g,h) [Cat.# L1170] Systems).

D. Renilla Luciferase Reporter Gene (Rluc)
The Renilla luciferase cDNA inserted into all of the pRL Vectors is derived from
the anthozoan coelentrate Renilla reniformis (1) but contains nucleotide changes
that were engineered during the construction of the individual vectors. The following
bases were altered in the pRL-null Vector: base 539 (T→C), to eliminate
an internal Bgl II site; base 1082 (T→C), to eliminate an internal BamH I site;
base 1115 (C→T), to eliminate internal Nar I, Kas I, Ban I and Acy I sites. These
nucleotide substitutions do not alter the amino acid sequence of the encoded
Renilla luciferase reporter enzyme.

E. SV40 Late Polyadenylation Signal
Polyadenylation signals cause the termination of transcription by RNA polymerase
II and signal the addition of approximately 200–250 adenosine residues
to the 3′-end of the RNA transcript (10). Polyadenylation has been shown to
enhance RNA stability and translation (11,12). The late SV40 polyadenylation
signal, which is extremely efficient and has been shown to increase the steadystate
level of RNA approximately 5-fold over the early SV40 polyadenylation signal
(13), has been positioned 3′ to the Rluc gene in the pRL-null Vector to
increase the level of Renilla luciferase expression.

IV. Transfection of Mammalian Cells with the pRL-null Vector

The pRL-null Vector, once it has been modified to contain appropriate genetic regulatory
domains, may be used in combination with any experimental reporter vector to
cotransfect mammalian cells. However, it is important to realize that trans effects
between promoters on cotransfected plasmids can potentially affect reporter gene
expression (14). Primarily this is of concern when either the control or experimental
reporter vector, or both, contain very strong promoter/enhancer elements. The
occurrence and magnitude of such effects will depend on several factors: i) the combination
and activities of the genetic regulatory elements present on the cotransfected
vectors; ii) the relative ratio of experimental vector to control vector introduced
into the cells; and iii) the cell type transfected.
To help ensure independent genetic expression between experimental and control
reporter genes, preliminary cotransfection experiments should be performed to optimize
both the amount of vector DNA and the ratio of the coreporter vectors added to
the transfection mixture. Similar to the firefly luciferase assay, the Renilla luciferase
assay is extremely sensitive, providing accurate measurement of ≤10 femtograms of
Renilla luciferase, with linearity over seven orders of enzyme concentration.
Therefore, it is possible to use relatively small quantities of the pRL-null Vector to
provide low-level, constitutive coexpression of Renilla luciferase control activity.
The pRL-null Vector, once genetic regulatory domains have been added, can be
used for both transient and stable expression of Renilla luciferase. For stable expression,
the pRL-null Vector must be cotransfected with an expression vector containing
a selectable gene in mammalian cells. Transfection of DNA into mammalian cells
may be mediated by cationic lipids (15,16), calcium phosphate (17,18), DEAEDextran
(19–21), polybrene-DMSO (22,23), or electroporation (24,25).
Transfection systems based on cationic lipid compounds (TransFast Reagent(i),
Tfx Reagents(j) and Transfectam Reagent(k)), calcium phosphate and DEAEDextran
are available from Promega. For more information and a protocol for the
Transfectam Reagent, please request the Transfectam Reagent Technical Bulletin
(#TB116) and for the TransFast Reagent, please request the TransFast
Transfection Reagent Technical Bulletin (#TB260). Protocols for the use of the
Tfx Reagents can be found in the Tfx-10, Tfx-20 and Tfx-50 Reagents for
the Transfection of Eukaryotic Cells Technical Bulletin (#TB216). For transfection procedures
using calcium phosphate or DEAE-Dextran, please request the ProFection
Mammalian Transfection Systems Technical Manual (#TM012).

载体序列

   1  AGATCTCGAG CTCTAAGCTT CACATATGCA TGCACTAGTG GCGCCTGTCG

  51  ACGCGTAGAA TTCACCCGGG TACTGCAGAA GTTGGTCGTG AGGCACTGGG

 101  CAGGTAAGTA TCAAGGTTAC AAGACAGGTT TAAGGAGACC AATAGAAACT

 151  GGGCTTGTCG AGACAGAGAA GACTCTTGCG TTTCTGATAG GCACCTATTG

 201  GTCTTACTGA CATCCACTTT GCCTTTCTCT CCACAGGTGT CCACTCCCAG

 251  TTCAATTACA GCTCTTAAGG CTAGAGTACT TAATACGACT CACTATAGGC

 301  TAGCCACCAT GACTTCGAAA GTTTATGATC CAGAACAAAG GAAACGGATG

 351  ATAACTGGTC CGCAGTGGTG GGCCAGATGT AAACAAATGA ATGTTCTTGA

 401  TTCATTTATT AATTATTATG ATTCAGAAAA ACATGCAGAA AATGCTGTTA

 451  TTTTTTTACA TGGTAACGCG GCCTCTTCTT ATTTATGGCG ACATGTTGTG

 501  CCACATATTG AGCCAGTAGC GCGGTGTATT ATACCAGACC TTATTGGTAT

 551  GGGCAAATCA GGCAAATCTG GTAATGGTTC TTATAGGTTA CTTGATCATT

 601  ACAAATATCT TACTGCATGG TTTGAACTTC TTAATTTACC AAAGAAGATC

 651  ATTTTTGTCG GCCATGATTG GGGTGCTTGT TTGGCATTTC ATTATAGCTA

 701  TGAGCATCAA GATAAGATCA AAGCAATAGT TCACGCTGAA AGTGTAGTAG

 751  ATGTGATTGA ATCATGGGAT GAATGGCCTG ATATTGAAGA AGATATTGCG

 801  TTGATCAAAT CTGAAGAAGG AGAAAAAATG GTTTTGGAGA ATAACTTCTT

 851  CGTGGAAACC ATGTTGCCAT CAAAAATCAT GAGAAAGTTA GAACCAGAAG

 901  AATTTGCAGC ATATCTTGAA CCATTCAAAG AGAAAGGTGA AGTTCGTCGT

 951  CCAACATTAT CATGGCCTCG TGAAATCCCG TTAGTAAAAG GTGGTAAACC

1001  TGACGTTGTA CAAATTGTTA GGAATTATAA TGCTTATCTA CGTGCAAGTG

1051  ATGATTTACC AAAAATGTTT ATTGAATCGG ACCCAGGATT CTTTTCCAAT

1101  GCTATTGTTG AAGGTGCCAA GAAGTTTCCT AATACTGAAT TTGTCAAAGT

1151  AAAAGGTCTT CATTTTTCGC AAGAAGATGC ACCTGATGAA ATGGGAAAAT

1201  ATATCAAATC GTTCGTTGAG CGAGTTCTCA AAAATGAACA ATAATTCTAG

1251  AGCGGCCGCT TCGAGCAGAC ATGATAAGAT ACATTGATGA GTTTGGACAA

1301  ACCACAACTA GAATGCAGTG AAAAAAATGC TTTATTTGTG AAATTTGTGA

1351  TGCTATTGCT TTATTTGTAA CCATTATAAG CTGCAATAAA CAAGTTAACA

1401  ACAACAATTG CATTCATTTT ATGTTTCAGG TTCAGGGGGA GGTGTGGGAG

1451  GTTTTTTAAA GCAAGTAAAA CCTCTACAAA TGTGGTAAAA TCGATAAGGA

1501  TCCAGGTGGC ACTTTTCGGG GAAATGTGCG CGGAACCCCT ATTTGTTTAT

1551  TTTTCTAAAT ACATTCAAAT ATGTATCCGC TCATGAGACA ATAACCCTGA

1601  TAAATGCTTC AATAATATTG AAAAAGGAAG AGTATGAGTA TTCAACATTT

1651  CCGTGTCGCC CTTATTCCCT TTTTTGCGGC ATTTTGCCTT CCTGTTTTTG

1701  CTCACCCAGA AACGCTGGTG AAAGTAAAAG ATGCTGAAGA TCAGTTGGGT

1751  GCACGAGTGG GTTACATCGA ACTGGATCTC AACAGCGGTA AGATCCTTGA

1801  GAGTTTTCGC CCCGAAGAAC GTTTTCCAAT GATGAGCACT TTTAAAGTTC

1851  TGCTATGTGG CGCGGTATTA TCCCGTATTG ACGCCGGGCA AGAGCAACTC

1901  GGTCGCCGCA TACACTATTC TCAGAATGAC TTGGTTGAGT ACTCACCAGT

1951  CACAGAAAAG CATCTTACGG ATGGCATGAC AGTAAGAGAA TTATGCAGTG

2001  CTGCCATAAC CATGAGTGAT AACACTGCGG CCAACTTACT TCTGACAACG

2051  ATCGGAGGAC CGAAGGAGCT AACCGCTTTT TTGCACAACA TGGGGGATCA

2101  TGTAACTCGC CTTGATCGTT GGGAACCGGA GCTGAATGAA GCCATACCAA

2151  ACGACGAGCG TGACACCACG ATGCCTGTAG CAATGGCAAC AACGTTGCGC

2201  AAACTATTAA CTGGCGAACT ACTTACTCTA GCTTCCCGGC AACAATTAAT

2251  AGACTGGATG GAGGCGGATA AAGTTGCAGG ACCACTTCTG CGCTCGGCCC

2301  TTCCGGCTGG CTGGTTTATT GCTGATAAAT CTGGAGCCGG TGAGCGTGGG

2351  TCTCGCGGTA TCATTGCAGC ACTGGGGCCA GATGGTAAGC CCTCCCGTAT

2401  CGTAGTTATC TACACGACGG GGAGTCAGGC AACTATGGAT GAACGAAATA

2451  GACAGATCGC TGAGATAGGT GCCTCACTGA TTAAGCATTG GTAACTGTCA

2501  GACCAAGTTT ACTCATATAT ACTTTAGATT GATTTAAAAC TTCATTTTTA

2551  ATTTAAAAGG ATCTAGGTGA AGATCCTTTT TGATAATCTC ATGACCAAAA

2601  TCCCTTAACG TGAGTTTTCG TTCCACTGAG CGTCAGACCC CGTAGAAAAG

2651  ATCAAAGGAT CTTCTTGAGA TCCTTTTTTT CTGCGCGTAA TCTGCTGCTT

2701  GCAAACAAAA AAACCACCGC TACCAGCGGT GGTTTGTTTG CCGGATCAAG

2751  AGCTACCAAC TCTTTTTCCG AAGGTAACTG GCTTCAGCAG AGCGCAGATA

2801  CCAAATACTG TTCTTCTAGT GTAGCCGTAG TTAGGCCACC ACTTCAAGAA

2851  CTCTGTAGCA CCGCCTACAT ACCTCGCTCT GCTAATCCTG TTACCAGTGG

2901  CTGCTGCCAG TGGCGATAAG TCGTGTCTTA CCGGGTTGGA CTCAAGACGA

2951  TAGTTACCGG ATAAGGCGCA GCGGTCGGGC TGAACGGGGG GTTCGTGCAC

3001  ACAGCCCAGC TTGGAGCGAA CGACCTACAC CGAACTGAGA TACCTACAGC

3051  GTGAGCTATG AGAAAGCGCC ACGCTTCCCG AAGGGAGAAA GGCGGACAGG

3101  TATCCGGTAA GCGGCAGGGT CGGAACAGGA GAGCGCACGA GGGAGCTTCC

3151  AGGGGGAAAC GCCTGGTATC TTTATAGTCC TGTCGGGTTT CGCCACCTCT

3201  GACTTGAGCG TCGATTTTTG TGATGCTCGT CAGGGGGGCG GAGCCTATGG

3251  AAAAACGCCA GCAACGCGGC CTTTTTACGG TTCCTGGCCT TTTGCTGGCC

3301  TTTTGCTCAC ATGGCTCGAC

载体质粒图谱pdf版和相关资料下载

载体应用举例

分享到:

全部载体分类

相关载体

相关文章

相关问答

Copyright © 20012-2013 BIOFENG. 生物风 版权所有 Powered by Biofeng