pCRE-MetLuc2-Reporter载体

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 expressionvector 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 productionrequires 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 E1References: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.

LOCUS       pCRE-MetLuc2-Reporter   4489 bp    DNA   circular             8-NOV-2009COMMENT     Created by Clontech Laboratories Inc. http://www.clontech.comCOMMENT     Cat. No. 631744,631745FEATURES             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\originBASE 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 //