pCRE-DD-tdTomato

pCRE-DD-tdTomato

pCRE-DD-tdTomato载体基本信息

pCRE-DD-tdTomato载体质粒图谱和多克隆位点信息

pCRE-DD-tdTomato载体描述

pCRE-DD-tdTomato is a reporter vector that allows you to monitor cAMP response element binding protein (CREB) activation. 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. The vector encodes the reporter protein DD-tdTomato, a ligand-dependent, red fluorescent protein that minimizes background fluorescence from leaky promoters. tdTomato is a member of the family of fruit fluorescent proteins derived from the Discosoma sp. red fluorescent protein, DsRed (excitation and emission maxima: 554 and 581 nm, respectively; 2, 3). DD-tdTomato is a modified version of tdTomato that is tagged on its N-terminus with the ProteoTuner destabilization domain (DD; 4). The presence of this destabilization domain causes rapid, proteasomal degradation of the fluorescent fusion protein; however, when the membrane permeant ligand Shield1 is added to the medium, it binds to the destabilization domain and protects the fusion protein from degradation. In the absence of Shield1, the destabilization domain causes the degradation of any DD-tdTomato reporter protein produced prior to promoter activation, thus reducing background fluorescence. In order to analyze CREB activation, an inducer of choice is added to the medium along with the Shield1 stabilizing ligand, which effectively stabilizes the reporter protein, allowing it to accumulate. As a result, only the reporter molecules expressed during promoter induction will contribute to the fluorescence signal, providing a considerably higher signal-to-noise ratio than that obtained with non-destabilized or constitutively destabilized reporter systems. The high signal-to-noise ratio also allows the monitoring of CREB activation during discrete windows of time when Shield1 is added to the cell medium for discrete periods of time.

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 (5). This cassette consists of the SV40 early promoter, a Tn5 kanamycin/neomycin resistance gene, and herpes simplex virus thymidine kinase (HSV TK) polyadenylation signals. A bacterial promoter upstream of the cassette expresses kanamycin resistance in E. coli.

The pCRE-DD-tdTomato Reporter vector, available as part of the CRE DD Red Reporter System (Cat. No. 631087), can be used to monitor CREB activation in live cells as well as in vivo. pCRE-DD-tdTomato can be transfected into mammalian cells using any standard transfection method. If required, stable transfectants can be selected using G418.

Propagation in E. coli

 Recommended 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

 Copy number: high

 Plasmid incompatibility group: pMB1/ColE1

Excitation and emission maxima of tdTomato

 Excitation maximum = 554 nm

 Emission maximum = 581 nm

References

1. Himmler, A. et al. (1993) J. Recep. Res. 13(1–4):79–94.

2. Shaner, N. C., et al. (2004) Nature Biotech. 22(12):1567-1572.

3. Campbell, R. E. et al. (2002) Proc. Natl. Acad. Sci. USA 99(12):7877–7882.

4. Banaszynski, L. et al. (2006) Cell 126(5):995–1004.

5. Gorman, C. (1985) In DNA Cloning: A Practical Approach, Vol. II. Ed. D. M. Glover. (IRL Press, Oxford, U.K.), pp. 143–190.

pCRE-DD-tdTomato载体序列

ORIGIN

        1 TAGTTATTAC TAGCGCTACC GGACTCAGAT CTCGAGCTCT TACGCGTGCT AGCGCACCAG

       61 ACAGTGACGT CAGCTGCCAG ATCCCATGGC CGTCATACTG TGACGTCTTT CAGACACCCC

      121 ATTGACGTCA ATGGGAGAAC AGATCTGCCG CCCCGACTGC ATCTGCGTGT TCGAATTCGC

      181 CAATGACAAG ACGCTGGGCG GGGTTTGTGT CATCATAGAA CTAAAGACAT GCAAATATAT

      241 TTCTTCCGGG GACACCGCCA GCAAACGCGA GCAACGGGCC ACGGGGATGA AGCAGAAGCT

      301 TCGAATTCTG CAGTCGACGG TACCGCGGGC CCGGGATCCA CCGGTCGCCA CCATGGGAGT

      361 GCAGGTGGAA ACCATCTCCC CAGGAGACGG GCGCACCTTC CCCAAGCGCG GCCAGACCTG

      421 TGTGGTGCAC TACACCGGGA TGCTTGAAGA TGGAAAGAAA GTCGATTCCT CCCGGGACAG

      481 AAACAAGCCC TTTAAGTTTA TGCTAGGCAA GCAGGAGGTG ATCCGAGGCT GGGAAGAAGG

      541 GGTTGCTCAG ATGAGTGTGG GTCAGAGAGC CAAACTGACT ATATCTCCAG ATTATGCCTA

      601 TGGTGCCACT GGGCACCCAG GCATCATCCC ACCACATGCC ACTCTCGTCT TCGATGTGGA

      661 GCTTCTAAAA CCGGAAAGAT CTATGGTGAG CAAGGGCGAG GAGGTCATCA AAGAGTTCAT

      721 GCGCTTCAAG GTGCGCATGG AGGGCTCCAT GAACGGCCAC GAGTTCGAGA TCGAGGGCGA

      781 GGGCGAGGGC CGCCCCTACG AGGGCACCCA GACCGCCAAG CTGAAGGTGA CCAAGGGCGG

      841 CCCCCTGCCC TTCGCCTGGG ACATCCTGTC CCCCCAGTTC ATGTACGGCT CCAAGGCGTA

      901 CGTGAAGCAC CCCGCCGACA TCCCCGATTA CAAGAAGCTG TCCTTCCCCG AGGGCTTCAA

      961 GTGGGAGCGC GTGATGAACT TCGAGGACGG CGGTCTGGTG ACCGTGACCC AGGACTCCTC

     1021 CCTGCAGGAC GGCACGCTGA TCTACAAGGT GAAGATGCGC GGCACCAACT TCCCCCCCGA

     1081 CGGCCCCGTA ATGCAGAAGA AGACCATGGG CTGGGAGGCC TCCACCGAGC GCCTGTACCC

     1141 CCGCGACGGC GTGCTGAAGG GCGAGATCCA CCAGGCCCTG AAGCTGAAGG ACGGCGGCCA

     1201 CTACCTGGTG GAGTTCAAGA CCATCTACAT GGCCAAGAAG CCCGTGCAAC TGCCCGGCTA

     1261 CTACTACGTG GACACCAAGC TGGACATCAC CTCCCACAAC GAGGACTACA CCATCGTGGA

     1321 ACAGTACGAG CGCTCCGAGG GCCGCCACCA CCTGTTCCTG GGGCATGGCA CCGGCAGCAC

     1381 CGGCAGCGGC AGCTCCGGCA CCGCCTCCTC CGAGGACAAC AACATGGCCG TCATCAAAGA

     1441 GTTCATGCGC TTCAAGGTGC GCATGGAGGG CTCCATGAAC GGCCACGAGT TCGAGATCGA

     1501 GGGCGAGGGC GAGGGCCGCC CCTACGAGGG CACCCAGACC GCCAAGCTGA AGGTGACCAA

     1561 GGGCGGCCCC CTGCCCTTCG CCTGGGACAT CCTGTCCCCC CAGTTCATGT ACGGCTCCAA

     1621 GGCGTACGTG AAGCACCCCG CCGACATCCC CGATTACAAG AAGCTGTCCT TCCCCGAGGG

     1681 CTTCAAGTGG GAGCGCGTGA TGAACTTCGA GGACGGCGGT CTGGTGACCG TGACCCAGGA

     1741 CTCCTCCCTG CAGGACGGCA CGCTGATCTA CAAGGTGAAG ATGCGCGGCA CCAACTTCCC

     1801 CCCCGACGGC CCCGTAATGC AGAAGAAGAC CATGGGCTGG GAGGCCTCCA CCGAGCGCCT

     1861 GTACCCCCGC GACGGCGTGC TGAAGGGCGA GATCCACCAG GCCCTGAAGC TGAAGGACGG

     1921 CGGCCACTAC CTGGTGGAGT TCAAGACCAT CTACATGGCC AAGAAGCCCG TGCAACTGCC

     1981 CGGCTACTAC TACGTGGACA CCAAGCTGGA CATCACCTCC CACAACGAGG ACTACACCAT

     2041 CGTGGAACAG TACGAGCGCT CCGAGGGCCG CCACCACCTG TTCCTGTACG GCATGGACGA

     2101 GCTGTACAAG TAGGCGGCCG CGACTCTAGA TCATAATCAG CCATACCACA TTTGTAGAGG

     2161 TTTTACTTGC TTTAAAAAAC CTCCCACACC TCCCCCTGAA CCTGAAACAT AAAATGAATG

     2221 CAATTGTTGT TGTTAACTTG TTTATTGCAG CTTATAATGG TTACAAATAA AGCAATAGCA

     2281 TCACAAATTT CACAAATAAA GCATTTTTTT CACTGCATTC TAGTTGTGGT TTGTCCAAAC

     2341 TCATCAATGT ATCTTAAGGC GTAAATTGTA AGCGTTAATA TTTTGTTAAA ATTCGCGTTA

     2401 AATTTTTGTT AAATCAGCTC ATTTTTTAAC CAATAGGCCG AAATCGGCAA AATCCCTTAT

     2461 AAATCAAAAG AATAGACCGA GATAGGGTTG AGTGTTGTTC CAGTTTGGAA CAAGAGTCCA

     2521 CTATTAAAGA ACGTGGACTC CAACGTCAAA GGGCGAAAAA CCGTCTATCA GGGCGATGGC

     2581 CCACTACGTG AACCATCACC CTAATCAAGT TTTTTGGGGT CGAGGTGCCG TAAAGCACTA

     2641 AATCGGAACC CTAAAGGGAG CCCCCGATTT AGAGCTTGAC GGGGAAAGCC GGCGAACGTG

     2701 GCGAGAAAGG AAGGGAAGAA AGCGAAAGGA GCGGGCGCTA GGGCGCTGGC AAGTGTAGCG

     2761 GTCACGCTGC GCGTAACCAC CACACCCGCC GCGCTTAATG CGCCGCTACA GGGCGCGTCA

     2821 GGTGGCACTT TTCGGGGAAA TGTGCGCGGA ACCCCTATTT GTTTATTTTT CTAAATACAT

     2881 TCAAATATGT ATCCGCTCAT GAGACAATAA CCCTGATAAA TGCTTCAATA ATATTGAAAA

     2941 AGGAAGAGTC CTGAGGCGGA AAGAACCAGC TGTGGAATGT GTGTCAGTTA GGGTGTGGAA

     3001 AGTCCCCAGG CTCCCCAGCA GGCAGAAGTA TGCAAAGCAT GCATCTCAAT TAGTCAGCAA

     3061 CCAGGTGTGG AAAGTCCCCA GGCTCCCCAG CAGGCAGAAG TATGCAAAGC ATGCATCTCA

     3121 ATTAGTCAGC AACCATAGTC CCGCCCCTAA CTCCGCCCAT CCCGCCCCTA ACTCCGCCCA

     3181 GTTCCGCCCA TTCTCCGCCC CATGGCTGAC TAATTTTTTT TATTTATGCA GAGGCCGAGG

     3241 CCGCCTCGGC CTCTGAGCTA TTCCAGAAGT AGTGAGGAGG CTTTTTTGGA GGCCTAGGCT

     3301 TTTGCAAAGA TCGATCAAGA GACAGGATGA GGATCGTTTC GCATGATTGA ACAAGATGGA

     3361 TTGCACGCAG GTTCTCCGGC CGCTTGGGTG GAGAGGCTAT TCGGCTATGA CTGGGCACAA

     3421 CAGACAATCG GCTGCTCTGA TGCCGCCGTG TTCCGGCTGT CAGCGCAGGG GCGCCCGGTT

     3481 CTTTTTGTCA AGACCGACCT GTCCGGTGCC CTGAATGAAC TGCAAGACGA GGCAGCGCGG

     3541 CTATCGTGGC TGGCCACGAC GGGCGTTCCT TGCGCAGCTG TGCTCGACGT TGTCACTGAA

     3601 GCGGGAAGGG ACTGGCTGCT ATTGGGCGAA GTGCCGGGGC AGGATCTCCT GTCATCTCAC

     3661 CTTGCTCCTG CCGAGAAAGT ATCCATCATG GCTGATGCAA TGCGGCGGCT GCATACGCTT

     3721 GATCCGGCTA CCTGCCCATT CGACCACCAA GCGAAACATC GCATCGAGCG AGCACGTACT

     3781 CGGATGGAAG CCGGTCTTGT CGATCAGGAT GATCTGGACG AAGAGCATCA GGGGCTCGCG

     3841 CCAGCCGAAC TGTTCGCCAG GCTCAAGGCG AGCATGCCCG ACGGCGAGGA TCTCGTCGTG

     3901 ACCCATGGCG ATGCCTGCTT GCCGAATATC ATGGTGGAAA ATGGCCGCTT TTCTGGATTC

     3961 ATCGACTGTG GCCGGCTGGG TGTGGCGGAC CGCTATCAGG ACATAGCGTT GGCTACCCGT

     4021 GATATTGCTG AAGAGCTTGG CGGCGAATGG GCTGACCGCT TCCTCGTGCT TTACGGTATC

     4081 GCCGCTCCCG ATTCGCAGCG CATCGCCTTC TATCGCCTTC TTGACGAGTT CTTCTGAGCG

     4141 GGACTCTGGG GTTCGAAATG ACCGACCAAG CGACGCCCAA CCTGCCATCA CGAGATTTCG

     4201 ATTCCACCGC CGCCTTCTAT GAAAGGTTGG GCTTCGGAAT CGTTTTCCGG GACGCCGGCT

     4261 GGATGATCCT CCAGCGCGGG GATCTCATGC TGGAGTTCTT CGCCCACCCT AGGGGGAGGC

     4321 TAACTGAAAC ACGGAAGGAG ACAATACCGG AAGGAACCCG CGCTATGACG GCAATAAAAA

     4381 GACAGAATAA AACGCACGGT GTTGGGTCGT TTGTTCATAA ACGCGGGGTT CGGTCCCAGG

     4441 GCTGGCACTC TGTCGATACC CCACCGAGAC CCCATTGGGG CCAATACGCC CGCGTTTCTT

     4501 CCTTTTCCCC ACCCCACCCC CCAAGTTCGG GTGAAGGCCC AGGGCTCGCA GCCAACGTCG

     4561 GGGCGGCAGG CCCTGCCATA GCCTCAGGTT ACTCATATAT ACTTTAGATT GATTTAAAAC

     4621 TTCATTTTTA ATTTAAAAGG ATCTAGGTGA AGATCCTTTT TGATAATCTC ATGACCAAAA

     4681 TCCCTTAACG TGAGTTTTCG TTCCACTGAG CGTCAGACCC CGTAGAAAAG ATCAAAGGAT

     4741 CTTCTTGAGA TCCTTTTTTT CTGCGCGTAA TCTGCTGCTT GCAAACAAAA AAACCACCGC

     4801 TACCAGCGGT GGTTTGTTTG CCGGATCAAG AGCTACCAAC TCTTTTTCCG AAGGTAACTG

     4861 GCTTCAGCAG AGCGCAGATA CCAAATACTG TTCTTCTAGT GTAGCCGTAG TTAGGCCACC

     4921 ACTTCAAGAA CTCTGTAGCA CCGCCTACAT ACCTCGCTCT GCTAATCCTG TTACCAGTGG

     4981 CTGCTGCCAG TGGCGATAAG TCGTGTCTTA CCGGGTTGGA CTCAAGACGA TAGTTACCGG

     5041 ATAAGGCGCA GCGGTCGGGC TGAACGGGGG GTTCGTGCAC ACAGCCCAGC TTGGAGCGAA

     5101 CGACCTACAC CGAACTGAGA TACCTACAGC GTGAGCTATG AGAAAGCGCC ACGCTTCCCG

     5161 AAGGGAGAAA GGCGGACAGG TATCCGGTAA GCGGCAGGGT CGGAACAGGA GAGCGCACGA

     5221 GGGAGCTTCC AGGGGGAAAC GCCTGGTATC TTTATAGTCC TGTCGGGTTT CGCCACCTCT

     5281 GACTTGAGCG TCGATTTTTG TGATGCTCGT CAGGGGGGCG GAGCCTATGG AAAAACGCCA

     5341 GCAACGCGGC CTTTTTACGG TTCCTGGCCT TTTGCTGGCC TTTTGCTCAC ATGTAGAGAA

     5401 ATGTTCTGGC ACCTGCACTT GCACTGGGGA CAGCCTATTT TGCTAGTTTG TTTTGTTTCG

     5461 TTTTGTTTTG ATGGAGAGCG TATGTTAGTA CTATCGATTC ACACAAAAAA CCAACACACA

     5521 GATGTAATGA AAATAAAGAT ATTTTATTAC ATGTTCTTTC CTGCGTTATC CCCTGATTCT

     5581 GTGGATAACC GTATTACCGC CATGCAT

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