pNFkB-DD-tdTomato

pNFkB-DD-tdTomato载体基本信息

pNFkB-DD-tdTomato载体质粒图谱和多克隆位点信息

pNFkB-DD-tdTomato载体描述

pNFkB-DD-tdTomato is a reporter vector that allows you to monitor NFkB activation in mammalian cells. The vector contains an NFkB enhancer element (composed of four tandem copies of the NFkB consensus binding sequence; 1) upstream of a minimal TA promoter (PTA), which consists of the TATA box from the herpes simplex virus thymidine kinase (HSV-TK) promoter. The vector encodes the reporter protein DD-tdTomato, a ligand-dependent, destabilized 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; 1, 2). DD-tdTomato is a modified version of tdTomato that is tagged on its N-terminus with the ProteoTuner? destabilization domain (DD; 3). 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 NFκB 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 NFκB 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 (4). 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 pNFkB-DD-tdTomato Reporter vector, available as part of the NFkB DD Red Reporter System (Cat. No. 631081), can be used to monitor NFkB activation in live cells as well as in vivo. pNFkB-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. Pessara, U. & Koch, N. (1990) Mol. Cell Biol. 10(8):4146–4154.

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.

Note: The vector sequence was 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.

pNFkB-DD-tdTomato载体序列

ORIGIN

        1 TAGTTATTAC TAGCGCTACC GGACTCAGAT CTCGAGCTCT TACGCGTGCT AGCGGGAATT

       61 TCCGGGAATT TCCGGGAATT TCCGGGAATT TCCAGATCTA GACTCTAGAG GGTATATAAT

      121 GGAAGCTCGA ATTCCAGCTT GGCATTCCGG TACTGTTGGT AAAAAGCTTC GAATTCTGCA

      181 GTCGACGGTA CCGCGGGCCC GGGATCCACC GGTCGCCACC ATGGGAGTGC AGGTGGAAAC

      241 CATCTCCCCA GGAGACGGGC GCACCTTCCC CAAGCGCGGC CAGACCTGTG TGGTGCACTA

      301 CACCGGGATG CTTGAAGATG GAAAGAAAGT CGATTCCTCC CGGGACAGAA ACAAGCCCTT

      361 TAAGTTTATG CTAGGCAAGC AGGAGGTGAT CCGAGGCTGG GAAGAAGGGG TTGCTCAGAT

      421 GAGTGTGGGT CAGAGAGCCA AACTGACTAT ATCTCCAGAT TATGCCTATG GTGCCACTGG

      481 GCACCCAGGC ATCATCCCAC CACATGCCAC TCTCGTCTTC GATGTGGAGC TTCTAAAACC

      541 GGAAAGATCT ATGGTGAGCA AGGGCGAGGA GGTCATCAAA GAGTTCATGC GCTTCAAGGT

      601 GCGCATGGAG GGCTCCATGA ACGGCCACGA GTTCGAGATC GAGGGCGAGG GCGAGGGCCG

      661 CCCCTACGAG GGCACCCAGA CCGCCAAGCT GAAGGTGACC AAGGGCGGCC CCCTGCCCTT

      721 CGCCTGGGAC ATCCTGTCCC CCCAGTTCAT GTACGGCTCC AAGGCGTACG TGAAGCACCC

      781 CGCCGACATC CCCGATTACA AGAAGCTGTC CTTCCCCGAG GGCTTCAAGT GGGAGCGCGT

      841 GATGAACTTC GAGGACGGCG GTCTGGTGAC CGTGACCCAG GACTCCTCCC TGCAGGACGG

      901 CACGCTGATC TACAAGGTGA AGATGCGCGG CACCAACTTC CCCCCCGACG GCCCCGTAAT

      961 GCAGAAGAAG ACCATGGGCT GGGAGGCCTC CACCGAGCGC CTGTACCCCC GCGACGGCGT

     1021 GCTGAAGGGC GAGATCCACC AGGCCCTGAA GCTGAAGGAC GGCGGCCACT ACCTGGTGGA

     1081 GTTCAAGACC ATCTACATGG CCAAGAAGCC CGTGCAACTG CCCGGCTACT ACTACGTGGA

     1141 CACCAAGCTG GACATCACCT CCCACAACGA GGACTACACC ATCGTGGAAC AGTACGAGCG

     1201 CTCCGAGGGC CGCCACCACC TGTTCCTGGG GCATGGCACC GGCAGCACCG GCAGCGGCAG

     1261 CTCCGGCACC GCCTCCTCCG AGGACAACAA CATGGCCGTC ATCAAAGAGT TCATGCGCTT

     1321 CAAGGTGCGC ATGGAGGGCT CCATGAACGG CCACGAGTTC GAGATCGAGG GCGAGGGCGA

     1381 GGGCCGCCCC TACGAGGGCA CCCAGACCGC CAAGCTGAAG GTGACCAAGG GCGGCCCCCT

     1441 GCCCTTCGCC TGGGACATCC TGTCCCCCCA GTTCATGTAC GGCTCCAAGG CGTACGTGAA

     1501 GCACCCCGCC GACATCCCCG ATTACAAGAA GCTGTCCTTC CCCGAGGGCT TCAAGTGGGA

     1561 GCGCGTGATG AACTTCGAGG ACGGCGGTCT GGTGACCGTG ACCCAGGACT CCTCCCTGCA

     1621 GGACGGCACG CTGATCTACA AGGTGAAGAT GCGCGGCACC AACTTCCCCC CCGACGGCCC

     1681 CGTAATGCAG AAGAAGACCA TGGGCTGGGA GGCCTCCACC GAGCGCCTGT ACCCCCGCGA

     1741 CGGCGTGCTG AAGGGCGAGA TCCACCAGGC CCTGAAGCTG AAGGACGGCG GCCACTACCT

     1801 GGTGGAGTTC AAGACCATCT ACATGGCCAA GAAGCCCGTG CAACTGCCCG GCTACTACTA

     1861 CGTGGACACC AAGCTGGACA TCACCTCCCA CAACGAGGAC TACACCATCG TGGAACAGTA

     1921 CGAGCGCTCC GAGGGCCGCC ACCACCTGTT CCTGTACGGC ATGGACGAGC TGTACAAGTA

     1981 GGCGGCCGCG ACTCTAGATC ATAATCAGCC ATACCACATT TGTAGAGGTT TTACTTGCTT

     2041 TAAAAAACCT CCCACACCTC CCCCTGAACC TGAAACATAA AATGAATGCA ATTGTTGTTG

     2101 TTAACTTGTT TATTGCAGCT TATAATGGTT ACAAATAAAG CAATAGCATC ACAAATTTCA

     2161 CAAATAAAGC ATTTTTTTCA CTGCATTCTA GTTGTGGTTT GTCCAAACTC ATCAATGTAT

     2221 CTTAAGGCGT AAATTGTAAG CGTTAATATT TTGTTAAAAT TCGCGTTAAA TTTTTGTTAA

     2281 ATCAGCTCAT TTTTTAACCA ATAGGCCGAA ATCGGCAAAA TCCCTTATAA ATCAAAAGAA

     2341 TAGACCGAGA TAGGGTTGAG TGTTGTTCCA GTTTGGAACA AGAGTCCACT ATTAAAGAAC

     2401 GTGGACTCCA ACGTCAAAGG GCGAAAAACC GTCTATCAGG GCGATGGCCC ACTACGTGAA

     2461 CCATCACCCT AATCAAGTTT TTTGGGGTCG AGGTGCCGTA AAGCACTAAA TCGGAACCCT

     2521 AAAGGGAGCC CCCGATTTAG AGCTTGACGG GGAAAGCCGG CGAACGTGGC GAGAAAGGAA

     2581 GGGAAGAAAG CGAAAGGAGC GGGCGCTAGG GCGCTGGCAA GTGTAGCGGT CACGCTGCGC

     2641 GTAACCACCA CACCCGCCGC GCTTAATGCG CCGCTACAGG GCGCGTCAGG TGGCACTTTT

     2701 CGGGGAAATG TGCGCGGAAC CCCTATTTGT TTATTTTTCT AAATACATTC AAATATGTAT

     2761 CCGCTCATGA GACAATAACC CTGATAAATG CTTCAATAAT ATTGAAAAAG GAAGAGTCCT

     2821 GAGGCGGAAA GAACCAGCTG TGGAATGTGT GTCAGTTAGG GTGTGGAAAG TCCCCAGGCT

     2881 CCCCAGCAGG CAGAAGTATG CAAAGCATGC ATCTCAATTA GTCAGCAACC AGGTGTGGAA

     2941 AGTCCCCAGG CTCCCCAGCA GGCAGAAGTA TGCAAAGCAT GCATCTCAAT TAGTCAGCAA

     3001 CCATAGTCCC GCCCCTAACT CCGCCCATCC CGCCCCTAAC TCCGCCCAGT TCCGCCCATT

     3061 CTCCGCCCCA TGGCTGACTA ATTTTTTTTA TTTATGCAGA GGCCGAGGCC GCCTCGGCCT

     3121 CTGAGCTATT CCAGAAGTAG TGAGGAGGCT TTTTTGGAGG CCTAGGCTTT TGCAAAGATC

     3181 GATCAAGAGA CAGGATGAGG ATCGTTTCGC ATGATTGAAC AAGATGGATT GCACGCAGGT

     3241 TCTCCGGCCG CTTGGGTGGA GAGGCTATTC GGCTATGACT GGGCACAACA GACAATCGGC

     3301 TGCTCTGATG CCGCCGTGTT CCGGCTGTCA GCGCAGGGGC GCCCGGTTCT TTTTGTCAAG

     3361 ACCGACCTGT CCGGTGCCCT GAATGAACTG CAAGACGAGG CAGCGCGGCT ATCGTGGCTG

     3421 GCCACGACGG GCGTTCCTTG CGCAGCTGTG CTCGACGTTG TCACTGAAGC GGGAAGGGAC

     3481 TGGCTGCTAT TGGGCGAAGT GCCGGGGCAG GATCTCCTGT CATCTCACCT TGCTCCTGCC

     3541 GAGAAAGTAT CCATCATGGC TGATGCAATG CGGCGGCTGC ATACGCTTGA TCCGGCTACC

     3601 TGCCCATTCG ACCACCAAGC GAAACATCGC ATCGAGCGAG CACGTACTCG GATGGAAGCC

     3661 GGTCTTGTCG ATCAGGATGA TCTGGACGAA GAGCATCAGG GGCTCGCGCC AGCCGAACTG

     3721 TTCGCCAGGC TCAAGGCGAG CATGCCCGAC GGCGAGGATC TCGTCGTGAC CCATGGCGAT

     3781 GCCTGCTTGC CGAATATCAT GGTGGAAAAT GGCCGCTTTT CTGGATTCAT CGACTGTGGC

     3841 CGGCTGGGTG TGGCGGACCG CTATCAGGAC ATAGCGTTGG CTACCCGTGA TATTGCTGAA

     3901 GAGCTTGGCG GCGAATGGGC TGACCGCTTC CTCGTGCTTT ACGGTATCGC CGCTCCCGAT

     3961 TCGCAGCGCA TCGCCTTCTA TCGCCTTCTT GACGAGTTCT TCTGAGCGGG ACTCTGGGGT

     4021 TCGAAATGAC CGACCAAGCG ACGCCCAACC TGCCATCACG AGATTTCGAT TCCACCGCCG

     4081 CCTTCTATGA AAGGTTGGGC TTCGGAATCG TTTTCCGGGA CGCCGGCTGG ATGATCCTCC

     4141 AGCGCGGGGA TCTCATGCTG GAGTTCTTCG CCCACCCTAG GGGGAGGCTA ACTGAAACAC

     4201 GGAAGGAGAC AATACCGGAA GGAACCCGCG CTATGACGGC AATAAAAAGA CAGAATAAAA

     4261 CGCACGGTGT TGGGTCGTTT GTTCATAAAC GCGGGGTTCG GTCCCAGGGC TGGCACTCTG

     4321 TCGATACCCC ACCGAGACCC CATTGGGGCC AATACGCCCG CGTTTCTTCC TTTTCCCCAC

     4381 CCCACCCCCC AAGTTCGGGT GAAGGCCCAG GGCTCGCAGC CAACGTCGGG GCGGCAGGCC

     4441 CTGCCATAGC CTCAGGTTAC TCATATATAC TTTAGATTGA TTTAAAACTT CATTTTTAAT

     4501 TTAAAAGGAT CTAGGTGAAG ATCCTTTTTG ATAATCTCAT GACCAAAATC CCTTAACGTG

     4561 AGTTTTCGTT CCACTGAGCG TCAGACCCCG TAGAAAAGAT CAAAGGATCT TCTTGAGATC

     4621 CTTTTTTTCT GCGCGTAATC TGCTGCTTGC AAACAAAAAA ACCACCGCTA CCAGCGGTGG

     4681 TTTGTTTGCC GGATCAAGAG CTACCAACTC TTTTTCCGAA GGTAACTGGC TTCAGCAGAG

     4741 CGCAGATACC AAATACTGTT CTTCTAGTGT AGCCGTAGTT AGGCCACCAC TTCAAGAACT

     4801 CTGTAGCACC GCCTACATAC CTCGCTCTGC TAATCCTGTT ACCAGTGGCT GCTGCCAGTG

     4861 GCGATAAGTC GTGTCTTACC GGGTTGGACT CAAGACGATA GTTACCGGAT AAGGCGCAGC

     4921 GGTCGGGCTG AACGGGGGGT TCGTGCACAC AGCCCAGCTT GGAGCGAACG ACCTACACCG

     4981 AACTGAGATA CCTACAGCGT GAGCTATGAG AAAGCGCCAC GCTTCCCGAA GGGAGAAAGG

     5041 CGGACAGGTA TCCGGTAAGC GGCAGGGTCG GAACAGGAGA GCGCACGAGG GAGCTTCCAG

     5101 GGGGAAACGC CTGGTATCTT TATAGTCCTG TCGGGTTTCG CCACCTCTGA CTTGAGCGTC

     5161 GATTTTTGTG ATGCTCGTCA GGGGGGCGGA GCCTATGGAA AAACGCCAGC AACGCGGCCT

     5221 TTTTACGGTT CCTGGCCTTT TGCTGGCCTT TTGCTCACAT GTAGAGAAAT GTTCTGGCAC

     5281 CTGCACTTGC ACTGGGGACA GCCTATTTTG CTAGTTTGTT TTGTTTCGTT TTGTTTTGAT

     5341 GGAGAGCGTA TGTTAGTACT ATCGATTCAC ACAAAAAACC AACACACAGA TGTAATGAAA

     5401 ATAAAGATAT TTTATTACAT GTTCTTTCCT GCGTTATCCC CTGATTCTGT GGATAACCGT

     5461 ATTACCGCCA TGCAT

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