pNFkB-DD-tdTomato

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载体基本信息

出品公司: Clontech
载体名称: pNFkB-DD-tdTomato
质粒类型: 哺乳动物细胞表达载体;荧光报告载体
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: TA
载体大小: 5475 bp
5' 测序引物及序列: --
3' 测序引物及序列: --
载体标签: DD tag (N-端)
载体抗性: Kanamycin.html' target='_blank'>卡那霉素
筛选标记: 新霉素(Neomycin)
克隆菌株: DH5α 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pNFkB-DD-tdTomato表达DD-tdTomato;
DD-tdTomato与tdTomato相比,N端融合了ProteoTuner destabilization domain (DD)去稳定结构域,导致tdTomato在细胞内很快被蛋白酶降解,降低了背景荧光,是研究启动子及其它顺式转录元件活性的理想报告基因。
产品目录号: 631081
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

载体质粒图谱和多克隆位点信息

pNFkB-DD-tdTomato载体图谱

pNFkB-DD-tdTomato 载体特征

载体简介

 
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.

载体序列

LOCUS       pNFkB-DD-tdTomato   5475 bp    DNA   circular            21-DEC-2009
COMMENT     Created by Clontech Laboratories Inc. http://www.clontech.com
COMMENT     Cat. No. 631082,631081
FEATURES             Location/Qualifiers
     enhancer        54..93
                     /label=NFkB\enhancer\element
     promoter        113..119
                     /label=TA\minimal\promoter
     CDS             3211..4005
                     /label=Kan(R)/Neo(R)
     rep_origin      complement(2231..2686)
                     /label=f1\origin
     rep_origin      3027..3165
                     /label=SV40\origin
     rep_origin      4590..5233
                     /label=pUC\origin
     CDS             221..544
                     /label=DD
     CDS             551..1978
                     /label=tdTomato
     misc_feature    1979..1981
                     /label=Stop\codon
     polyA_signal    2134..2168
                     /label=SV40\early\polyA\signals
     polyA_signal    4241..4259
                     /label=HSV\TK\polyA\signals
     misc_signal     214..224
                     /label=Kozak\sequence
BASE COUNT     1298 a      1512 c      1495 g      1170 t 
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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