pMetLuc2-Reporter

pMetLuc2-Reporter

pMetLuc2-Reporter载体基本信息：

pMetLuc2-Reporter载体质粒图谱和多克隆位点信息：

pMetLuc2-Reporter载体描述：

pMetLuc2-Reporter is a promoter reporter vector that allows the analysis of promoter function in cell-based assays. The vector encodes a sequence-optimized, secreted luciferase from the marine copepod Metridia longa. The 24 kDa Metridia luciferase (MetLuc) reporter protein

contains a 17 amino acid, N-terminal signal peptide that allows efficient secretion of the reporter (1). When a functional promoter is cloned into the MCS, located upstream of the MetLuc reporter gene, MetLuc is expressed and secreted into the medium surrounding the transfected cells.

SV40 polyadenylation signals downstream of the MetLuc gene direct proper processing of the 3' end of the MetLuc mRNA. The vector backbone contains an SV40 origin for replication in mammalian cells expressing the SV40 large T antigen, a pUC origin of replication for propagation in E. coli, and an f1 origin for single-stranded DNA production. A neomycin resistance cassette (Neor) allows stably transfected eukaryotic cells to be selected using G418. This cassette consists of the SV40 early promoter, the Tn5 kanamycin/neomycin resistance gene, and polyadenylation signals from the herpes simplex virus thymidine kinase (HSV TK) gene. The vector also contains a synthetic transcription blocker (TB), composed of adjacent polyadenylation and transcription pause sites, that reduces background readthrough transcription (2). A bacterial promoter (PKanr) upstream of the cassette allows kanamycin resistance in E. coli.

The pMetLuc2-Reporter vector is used to monitor the activity of promoters cloned into the MCS, located upstream of the Metridia longa luciferase coding sequence. Upon induction, functional promoters will drive the expression of secreted luciferase, while nonfunctional promoters will not. The presence of the luciferase can be easily detected by adding luciferase substrate to a small aliquot of the culture medium and analyzing the sample in a luminometer. Promoter function can be quantified by the relative intensity of the bioluminescent signal.

The pMetLuc2-Reporter Vector can be transfected into mammalian cells using any standard transfection method. Stable transfectants can be selected using G418 when required (3).

Propagation in E. coli

 Suitable host strains: DH5α, HB101, and other general purpose strains. Single-stranded DNA production requires

a host containing an F plasmid such as JM109 or XL1-Blue.

 Selectable marker: plasmid confers resistance to kanamycin (50 μg/ml) in E. coli hosts.

 E. coli replication origin: pUC

 Plasmid incompatibility group: pMB1/Col E1

Note: The attached sequence file has been compiled from information in the sequence databases, published literature, and other sources, together with partial sequences obtained by Clontech. This vector has not been completely sequenced.

References

1. Markova, S.V. et al. (2004) J. Bio. Chem. 279(5):3212-3217.

2. Eggermont, J. & Proudfoot, N. (1993) EMBO J. 12(6):2539–2548.

3. Gorman, C. (1985) DNA cloning: A Practical Approach, Vol. II. Ed. D. M. Glover. (IRL Press Oxford, U.K.), pp. 143-190.

pMetLuc2-Reporter载体序列：

ORIGIN

        1 tagttattac tagcgctacc ggactcagat ctcgagctca agcttcgaat tctgcagtcg

       61 acggtaccgc gggcccggga tccaccggtc gccaccatgg acatcaaggt ggtgttcacc

      121 ctggtgttca gcgccctggt gcaggccaag agcaccgagt tcgaccccaa catcgacatc

      181 gtgggcctgg aaggcaagtt cggcatcacc aacctggaaa ccgacctgtt caccatctgg

      241 gagaccatgg aagtgatgat caaggccgac atcgccgaca ccgaccgggc cagcaacttc

      301 gtggccaccg agaccgacgc caaccggggc aagatgcccg gcaagaagct gcccctggcc

      361 gtcatcatgg aaatggaagc caacgccttc aaggccggct gcacccgggg ctgcctgatc

      421 tgcctgagca agatcaagtg caccgccaag atgaaggtgt acatccccgg caggtgccac

      481 gactacggcg gcgacaagaa aaccggccag gccggcatcg tgggcgccat cgtggacatc

      541 cccgagatca gcggcttcaa agaaatggcc cccatggaac agttcatcgc ccaggtggac

      601 agatgcgcca gctgcaccac cggctgcctg aagggcctgg ccaacgtgaa gtgcagcgag

      661 ctgctgaaga agtggctgcc cgaccgctgc gccagcttcg ccgacaagat ccagaaagag

      721 gtgcacaaca tcaagggcat ggccggcgac aggtgagcgg ccgcgactct agatcataat

      781 cagccatacc acatttgtag aggttttact tgctttaaaa aacctcccac acctccccct

      841 gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg cagcttataa

      901 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca

      961 ttctagttgt ggtttgtcca aactcatcaa tgtatcttaa ggcgtaaatt gtaagcgtta

     1021 atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg

     1081 ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg

     1141 ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa

     1201 aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg

     1261 ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt

     1321 gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg

     1381 ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta

     1441 atgcgccgct acagggcgcg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta

     1501 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat

     1561 aaatgcttca ataatattga aaaaggaaga gtcctgaggc ggaaagaacc agctgtggaa

     1621 tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag

     1681 catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag

     1741 aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc

     1801 catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt

     1861 ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg

     1921 aggctttttt ggaggcctag gcttttgcaa agatcgatca agagacagga tgaggatcgt

     1981 ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc

     2041 tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc

     2101 tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg

     2161 aactgcaaga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag

     2221 ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg

     2281 ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg

     2341 caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac

     2401 atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg

     2461 acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgagcatgc

     2521 ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg

     2581 aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc

     2641 aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc

     2701 gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc

     2761 ttcttgacga gttcttctga gcgggactct ggggttcgaa atgaccgacc aagcgacgcc

     2821 caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg

     2881 aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt

     2941 cttcgcccac cctaggggga ggctaactga aacacggaag gagacaatac cggaaggaac

     3001 ccgcgctatg acggcaataa aaagacagaa taaaacgcac ggtgttgggt cgtttgttca

     3061 taaacgcggg gttcggtccc agggctggca ctctgtcgat accccaccga gaccccattg

     3121 gggccaatac gcccgcgttt cttccttttc cccaccccac cccccaagtt cgggtgaagg

     3181 cccagggctc gcagccaacg tcggggcggc aggccctgcc atagcctcag gttactcata

     3241 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct

     3301 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga

     3361 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg

     3421 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc

     3481 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct

     3541 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc

     3601 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt

     3661 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg

     3721 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct

     3781 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag

     3841 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag

     3901 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg

     3961 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg

     4021 gccttttgct cacatgtaat aaaatatctt tattttcatt acatctgtgt gttggttttt

     4081 tgtgtgaatc gatagtacta acatacgctc tccatcaaaa caaaacgaaa caaaacaaac

     4141 tagcaaaata ggctgtcccc agtgcaagtg caggtgccag aacatttctc tacatgttct

     4201 ttcctgcgtt atcccctgat tctgtggata accgtattac cgccatgcat

//

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