| 出品公司: | Clontech |
|---|---|
| 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。 |
| 产品目录号: | 631729 & 631735 |
| 稳定性: | 瞬表达 或 稳表达 |
| 组成型/诱导型: | -- |
| 病毒/非病毒: | 非病毒 |
买家导航
载体基本信息
载体图谱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
//
