| 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: | 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 |
买家导航
pNFkB-DD-tdTomato载体质粒基本信息
pNFkB-DD-tdTomato质粒图谱载体图谱和pNFkB-DD-tdTomato载体序列质粒序列多克隆位点信息
pNFkB-DD-tdTomato质粒载体简介
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.
pNFkB-DD-tdTomato质粒序列载体序列
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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