| 出品公司: | Clontech |
|---|---|
| 载体名称: | pCRE-MetLuc2-Reporter |
| 质粒类型: | 哺乳动物细胞表达载体；信号通路报告载体；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: | TAL (TATA-like promoter） |
| 载体大小: | 4489 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）: | 常规细胞系（293、CV-1、CHO等） |
| 备注: |
哺乳动物细胞表达载体pCRE-MetLuc2-Reporter是荧光素酶报告载体； 用于研究cAMP介导的信号通路； MetLuc是已知分子量最小的荧光素酶； 荧光素酶报告基因既可以定性又可以定量。 |
| 产品目录号: | 631745 |
| 稳定性: | 瞬表达 或 稳表达 |
| 组成型/诱导型: | 组成型 |
| 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
pCRE-MetLuc2-Reporter载体描述
pCRE-MetLuc2-Reporter allows you to monitor cAMP-mediated signal transduction directly from the cell culture medium, without cell lysis. The vector contains two copies of the cAMP response element (CRE; 1) fused to a TATA-like promoter (PTAL) region from the herpes simplex virus thymidine kinase (HSV-TK) gene. Located downstream of PTAL 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, such as the cAMP response element binding protein (CREB), to the CRE sequence allows Metridia luciferase (MetLuc) to be expressed and secreted into the surrounding medium (2).
To prevent read-through transcription of the Metridia luciferase gene, a synthetic transcription blocker (TB; 3, 4), composed of adjacent polyadenylation and transcription pause sites, is located upstream of the CRE. 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.
pCRE-MetLuc2-Reporter is available as part of the Ready-To-Glow CRE Secreted Luciferase Reporter System (Cat. No. 631745). In mammalian cells containing pCRE-MetLuc2-Reporter, the addition of cAMP, or forskolin to the culture medium induces transcription factors, such as CREB or activating transcription factor (ATF) proteins, to bind to the CRE, thereby initiating transcription of the secreted luciferase reporter gene. Activation of cAMP-mediated signal transduction can be monitored simply by sampling the culture medium. To monitor the effect of a specific protein on the system, pCRE-MetLuc2-Reporter can be cotransfected with an expression
vector containing the gene of interest. pCRE-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 or 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. Himmler, A., et al. (1993) J. Recep. Res. 13(1–4):79–94.
2. Markova, S. V., et al. (2004) J. Biol. Chem. 279(5):3212–3217.
3. Eggermont, J. & Proudfoot, N. (1993) EMBO J. 12(6):2539–2548.
4. Enriquez-Harris, P., et al. (1991) EMBO J. 10(7):1833–1842.
Note:
The vector sequence file has been compiled from information in the sequence databases, published literature, and other sources,
together with partial sequences obtained by Clontech Laboratories, Inc. This vector has not been completely sequenced.
载体序列
LOCUS pCRE-MetLuc2-Reporter 4489 bp DNA circular 8-NOV-2009
COMMENT Created by Clontech Laboratories Inc. http://www.clontech.com
COMMENT Cat. No. 631744,631745
FEATURES Location/Qualifiers
misc_feature 37..123
/label=cAMP\response\element\(CRE)
promoter 130..278
/label=TATA\like\promoter\Ptal
CDS 336..992
/label=Metridia\secreted\luciferase
CDS 2225..3019
/label=Kan(R)/Neo(R)
misc_feature 4277..4430
/label=Transcription\blocker
polyA_signal 1148..1153
/label=SV40\early\polyA\signal
rep_origin complement(1245..1700)
/label=f1\origin
rep_origin 2041..2179
/label=SV40\origin
rep_origin 3604..4247
/label=pUC\origin
BASE COUNT 1097 a 1207 c 1193 g 992 t
ORIGIN
1 tagttattac tagcgctacc ggactcagat ctcgaggcac cagacagtga cgtcagctgc
61 cagatcccat ggccgtcata ctgtgacgtc tttcagacac cccattgacg tcaatgggag
121 aacagatctg ccgccccgac tgcatctgcg tgttcgaatt cgccaatgac aagacgctgg
181 gcggggtttg tgtcatcata gaactaaaga catgcaaata tatttcttcc ggggacaccg
241 ccagcaaacg cgagcaacgg gccacgggga tgaagcagaa gcttcgaatt ctgcagtcga
301 cggtaccgcg ggcccgggat ccaccggtcg ccaccatgga catcaaggtg gtgttcaccc
361 tggtgttcag cgccctggtg caggccaaga gcaccgagtt cgaccccaac atcgacatcg
421 tgggcctgga aggcaagttc ggcatcacca acctggaaac cgacctgttc accatctggg
481 agaccatgga agtgatgatc aaggccgaca tcgccgacac cgaccgggcc agcaacttcg
541 tggccaccga gaccgacgcc aaccggggca agatgcccgg caagaagctg cccctggccg
601 tcatcatgga aatggaagcc aacgccttca aggccggctg cacccggggc tgcctgatct
661 gcctgagcaa gatcaagtgc accgccaaga tgaaggtgta catccccggc aggtgccacg
721 actacggcgg cgacaagaaa accggccagg ccggcatcgt gggcgccatc gtggacatcc
781 ccgagatcag cggcttcaaa gaaatggccc ccatggaaca gttcatcgcc caggtggaca
841 gatgcgccag ctgcaccacc ggctgcctga agggcctggc caacgtgaag tgcagcgagc
901 tgctgaagaa gtggctgccc gaccgctgcg ccagcttcgc cgacaagatc cagaaagagg
961 tgcacaacat caagggcatg gccggcgaca ggtgagcggc cgcgactcta gatcataatc
1021 agccatacca catttgtaga ggttttactt gctttaaaaa acctcccaca cctccccctg
1081 aacctgaaac ataaaatgaa tgcaattgtt gttgttaact tgtttattgc agcttataat
1141 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
1201 tctagttgtg gtttgtccaa actcatcaat gtatcttaag gcgtaaattg taagcgttaa
1261 tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc
1321 cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt
1381 tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa
1441 aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg
1501 gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg
1561 acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc
1621 tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa
1681 tgcgccgcta cagggcgcgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
1741 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
1801 aatgcttcaa taatattgaa aaaggaagag tcctgaggcg gaaagaacca gctgtggaat
1861 gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
1921 atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga
1981 agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
2041 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
2101 tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga
2161 ggcttttttg gaggcctagg cttttgcaaa gatcgatcaa gagacaggat gaggatcgtt
2221 tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct
2281 attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct
2341 gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga
2401 actgcaagac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc
2461 tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg
2521 gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc
2581 aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca
2641 tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga
2701 cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg cgagcatgcc
2761 cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga
2821 aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca
2881 ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg
2941 cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct
3001 tcttgacgag ttcttctgag cgggactctg gggttcgaaa tgaccgacca agcgacgccc
3061 aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga
3121 atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc
3181 ttcgcccacc ctagggggag gctaactgaa acacggaagg agacaatacc ggaaggaacc
3241 cgcgctatga cggcaataaa aagacagaat aaaacgcacg gtgttgggtc gtttgttcat
3301 aaacgcgggg ttcggtccca gggctggcac tctgtcgata ccccaccgag accccattgg
3361 ggccaatacg cccgcgtttc ttccttttcc ccaccccacc ccccaagttc gggtgaaggc
3421 ccagggctcg cagccaacgt cggggcggca ggccctgcca tagcctcagg ttactcatat
3481 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
3541 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
3601 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
3661 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
3721 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
3781 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
3841 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
3901 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
3961 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
4021 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
4081 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
4141 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
4201 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
4261 ccttttgctc acatgtaata aaatatcttt attttcatta catctgtgtg ttggtttttt
4321 gtgtgaatcg atagtactaa catacgctct ccatcaaaac aaaacgaaac aaaacaaact
4381 agcaaaatag gctgtcccca gtgcaagtgc aggtgccaga acatttctct acatgttctt
4441 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gccatgcat
//
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