pMetLuc2-Reporter

pMetLuc2-Reporter

载体基本信息

出品公司:	Ybscience
载体名称:	pMetLuc2-Reporter
质粒类型:	无启动子载体；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:	无
载体大小:	4250 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:	DH5alpha等
宿主细胞（系）:	真核细胞
备注:	pMetLuc2-Reporter没有启动子元件； MetLuc是已知最小分子量的荧光素酶； 荧光素酶报告基因可以用来定性定量分析启动子及其它顺式转录元件的活性。
稳定性:	瞬表达 或 稳表达
组成型/诱导型:	--
病毒/非病毒:	非病毒

 

载体质粒图谱和多克隆位点信息

载体简介

 

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.

 

载体序列

LOCUS       pMetLuc2-Reporter   4250 bp    DNA   circular             8-NOV-2009
COMMENT     Created by Clontech Laboratories Inc. http://www.clontech.com
COMMENT     Cat. No. 631731,631729,631732,631735,631730
FEATURES             Location/Qualifiers
     misc_feature    27..89
                     /label=MCS
     CDS             97..753
                     /label=Metridia\secreted\luciferase
     CDS             1986..2780
                     /label=Kan(R)/Neo(R)
     misc_feature    4038..4191
                     /label=Transcription\blocker
     polyA_signal    909..914
                     /label=SV40\early\polyA\signal
     polyA_signal    938..943
                     /label=SV40\early\polyA\signal
     polyA_signal    3016..3021
                     /label=HSV\TK\polyA\signal
     polyA_signal    3029..3034
                     /label=HSV\TK\polyA\signal
     rep_origin      complement(1006..1461)
                     /label=f1\origin
     rep_origin      1802..1940
                     /label=SV40\origin
     rep_origin      3365..4008
                     /label=pUC\origin
     misc_feature    1857..1863
                     /label=SV40\early\promoter
     promoter        1523..1551
                     /label=Kan(R)\Promoter
BASE COUNT     1034 a      1141 c      1128 g       947 t 
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