pNFκB-MetLuc2-Reporter

pNFκB-MetLuc2-Reporter

pNFκB-MetLuc2-Reporter载体基本信息

pNFκB-MetLuc2-Reporter载体质粒图谱和多克隆位点信息

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载体序列

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

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