pCRE-MetLuc2-Reporter

pCRE-MetLuc2-Reporter载体基本信息

pCRE-MetLuc2-Reporter载体质粒图谱和多克隆位点信息

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.

pCRE-MetLuc2-Reporter载体序列

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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