| 出品公司: | Clontech |
|---|---|
| 载体名称: | pNFκB-MetLuc2-Reporter |
| 质粒类型: | 哺乳动物细胞表达载体；荧光素酶报告载体 |
| 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: | TA |
| 载体大小: | 4366 bp |
| 5' 测序引物及序列: | -- |
| 3' 测序引物及序列: | -- |
| 载体标签: | -- |
| 载体抗性: | 卡那霉素 |
| 筛选标记: | 新霉素（Neomycin） |
| 克隆菌株: | Stbl3 |
| 宿主细胞（系）: | 常规细胞系（293、CV-1、CHO等） |
| 备注: |
哺乳动物表达载体pNFκB-MetLuc2-Reporter是荧光素酶报告载体； MetLuc是已知分子量最小的荧光素酶； 启动子上游有NFκB增强子元件。 |
| 产品目录号: | 631743 |
| 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 |
买家导航
pNFκB-MetLuc2-Reporter载体质粒基本信息
pNFκB-MetLuc2-Reporter质粒图谱载体图谱和pNFκB-MetLuc2-Reporter载体序列质粒序列多克隆位点信息
pNFκB-MetLuc2-Reporter质粒载体简介
pNFκB-MetLuc2-Reporter载体描述
pNFκB-MetLuc2-Reporter allows you to monitor the activation of the NFκB signal transduction pathway (1–4) directly from the cell culture medium, without cell lysis. The vector contains an NFκB enhancer element (composed of four tandem copies of the NFκB consensus binding sequence; 5), located upstream of the minimal TA promoter (PTA), which consists of the TATA box from the herpes simplex virus thymidine kinase (HSV-TK) promoter. Located downstream of PTA is a human codon optimized secreted-luciferase gene from the marine copepod Metridia longa. This gene encodes a 219 amino acid (24 kDa) polypeptide that includes a 17 amino acid N-terminal signal peptide necessary for secretion. Binding of transcription factors to the NFκB enhancer element allows Metridia luciferase (MetLuc) to be expressed and secreted into the surrounding medium (6).
To prevent read-through transcription of the Metridia luciferase gene, a synthetic transcription
blocker (TB; 7, 8), composed of adjacent polyadenylation and transcription pause sites, is located upstream of the cis-acting enhancer element. To ensure efficient processing of the luciferase transcript in eukaryotic cells, an SV40 early polyadenylation signal is located downstream of the Metridia luciferase coding sequence. The vector backbone also contains an f1 origin for single-stranded DNA production, a pUC origin of replication, and a kanamycin resistance gene for propagation and selection in E. coli.
pNFκB-MetLuc2-Reporter is available as part of the Ready-To-Glow? NFκB Secreted Luciferase Reporter
System (Cat. No. 631743). In mammalian cells containing pNFκB-MetLuc2-Reporter, the addition of TNFα, IL-1, or stimulants of lymphokine receptors to the culture medium induces transcription factors to bind to the NFκB enhancer element, thereby initiating transcription of the secreted luciferase reporter gene.
Activation of the NFκB signal transduction pathway can be monitored simply by sampling the culture medium. To monitor the effect of a specific protein on the pathway, pNFκB-MetLuc2-Reporter can be cotransfected with an expression vector containing the gene of interest. pNFkB-MetLuc2-Reporter can be transfected into mammalian cells using any standard transfection method. Stable transfectants can be selected using G418 when required.
Propagation in E. coli:
Suitable host strains: DH5α?, HB101, and other general purpose strains. Single-stranded DNA production
requires a host containing an F' episome such as JM109 and XL1-Blue.
Selectable marker: plasmid confers resistance to kanamycin (50 μg/ml) to E. coli hosts.
E. coli replication origin: pUC
Plasmid incompatibility group: pMB1/Col E1
References:
1. NF-κB Transcription Reporter Vectors (July 1998) Clontechniques XIII(3):24–25.
2. Baeuerle, P. A. & Baltimore, D. (1996) Cell 87(1):13–20
3. Baeuerle, P. A. (1998) Curr. Biol. 8(1):R19–R22.
4. Peltz, G. (1997) Curr. Opin. Biotechnol. 8(4):467–473.
5. Pessara, U. & Koch, N. (1990) Mol. Cell Biol. 10(8):4146–4154.
6. Markova, S. V., et al. (2004) J. Biol. Chem. 279(5):3212–3217..
7. Eggermont, J. & Proudfoot, N. (1993) EMBO J. 12(6):2539–2548.
8. Enriquez-Harris, P., et al. (1991) EMBO J. 10(7):1833–1842.
pNFκB-MetLuc2-Reporter质粒序列载体序列
LOCUS pNFkB-MetLuc2-Reporter 4366 bp DNA circular 8-NOV-2009
COMMENT Created by Clontech Laboratories Inc. http://www.clontech.com
COMMENT Cat. No. 631742,631743
FEATURES Location/Qualifiers
enhancer 46..85
/label=NFkB\enhancer\element
promoter 105..111
/label=TA\minimal\promoter
CDS 213..869
/label=Metridia\secreted\luciferase
CDS 2102..2896
/label=Kan(R)/Neo(R)
misc_feature 4154..4307
/label=Transcription\blocker
polyA_signal 1025..1030
/label=SV40\early\polyA\signal
rep_origin complement(1122..1577)
/label=f1\origin
rep_origin 1918..2056
/label=SV40\origin
rep_origin 3481..4124
/label=pUC\origin
BASE COUNT 1064 a 1162 c 1159 g 981 t
ORIGIN
1 tagttattac tagcgctacc ggactcagat ctcgagctca agcttgggaa tttccgggaa
61 tttccgggaa tttccgggaa tttccagatc tagactctag agggtatata atggaagctc
121 gaattccagc ttggcattcc ggtactgttg gtaaaaagct tcgaattctg cagtcgacgg
181 taccgcgggc ccgggatcca ccggtcgcca ccatggacat caaggtggtg ttcaccctgg
241 tgttcagcgc cctggtgcag gccaagagca ccgagttcga ccccaacatc gacatcgtgg
301 gcctggaagg caagttcggc atcaccaacc tggaaaccga cctgttcacc atctgggaga
361 ccatggaagt gatgatcaag gccgacatcg ccgacaccga ccgggccagc aacttcgtgg
421 ccaccgagac cgacgccaac cggggcaaga tgcccggcaa gaagctgccc ctggccgtca
481 tcatggaaat ggaagccaac gccttcaagg ccggctgcac ccggggctgc ctgatctgcc
541 tgagcaagat caagtgcacc gccaagatga aggtgtacat ccccggcagg tgccacgact
601 acggcggcga caagaaaacc ggccaggccg gcatcgtggg cgccatcgtg gacatccccg
661 agatcagcgg cttcaaagaa atggccccca tggaacagtt catcgcccag gtggacagat
721 gcgccagctg caccaccggc tgcctgaagg gcctggccaa cgtgaagtgc agcgagctgc
781 tgaagaagtg gctgcccgac cgctgcgcca gcttcgccga caagatccag aaagaggtgc
841 acaacatcaa gggcatggcc ggcgacaggt gagcggccgc gactctagat cataatcagc
901 cataccacat ttgtagaggt tttacttgct ttaaaaaacc tcccacacct ccccctgaac
961 ctgaaacata aaatgaatgc aattgttgtt gttaacttgt ttattgcagc ttataatggt
1021 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
1081 agttgtggtt tgtccaaact catcaatgta tcttaaggcg taaattgtaa gcgttaatat
1141 tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga
1201 aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc
1261 agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac
1321 cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc
1381 gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg
1441 gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag
1501 ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc
1561 gccgctacag ggcgcgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
1621 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
1681 gcttcaataa tattgaaaaa ggaagagtcc tgaggcggaa agaaccagct gtggaatgtg
1741 tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
1801 catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt
1861 atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc
1921 ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt
1981 atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc
2041 ttttttggag gcctaggctt ttgcaaagat cgatcaagag acaggatgag gatcgtttcg
2101 catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt
2161 cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc
2221 agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact
2281 gcaagacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt
2341 gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca
2401 ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat
2461 gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg
2521 catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga
2581 agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcga gcatgcccga
2641 cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa
2701 tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga
2761 catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt
2821 cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct
2881 tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac
2941 ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc
3001 gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc
3061 gcccacccta gggggaggct aactgaaaca cggaaggaga caataccgga aggaacccgc
3121 gctatgacgg caataaaaag acagaataaa acgcacggtg ttgggtcgtt tgttcataaa
3181 cgcggggttc ggtcccaggg ctggcactct gtcgataccc caccgagacc ccattggggc
3241 caatacgccc gcgtttcttc cttttcccca ccccaccccc caagttcggg tgaaggccca
3301 gggctcgcag ccaacgtcgg ggcggcaggc cctgccatag cctcaggtta ctcatatata
3361 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
3421 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
3481 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
3541 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
3601 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
3661 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
3721 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
3781 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
3841 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
3901 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
3961 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
4021 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
4081 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
4141 tttgctcaca tgtaataaaa tatctttatt ttcattacat ctgtgtgttg gttttttgtg
4201 tgaatcgata gtactaacat acgctctcca tcaaaacaaa acgaaacaaa acaaactagc
4261 aaaataggct gtccccagtg caagtgcagg tgccagaaca tttctctaca tgttctttcc
4321 tgcgttatcc cctgattctg tggataaccg tattaccgcc atgcat
//
