pNFκB-MetLuc2-Reporter载体

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 transcriptionblocker (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 ReporterSystem (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 productionrequires 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 E1References:1. NF-κB Transcription Reporter Vectors (July 1998) Clontechniques XIII(3):24–25.2. Baeuerle, P. A. & Baltimore, D. (1996) Cell 87(1):13–203. 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.

LOCUS       pNFkB-MetLuc2-Reporter   4366 bp    DNA   circular             8-NOV-2009COMMENT     Created by Clontech Laboratories Inc. http://www.clontech.comCOMMENT     Cat. No. 631742,631743FEATURES             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\originBASE 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