| 出品公司: | Clontech |
|---|---|
| 载体名称: | pCRE-DD-tdTomato |
| 质粒类型: | 哺乳动物细胞表达载体；信号通路报告载体 |
| 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: | TAL |
| 载体大小: | 5607 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, HB101 |
| 宿主细胞（系）: | 常规细胞系（293、CV-1、CHO等） |
| 备注: |
哺乳动物载体pCRE-DD-tdTomato是cAMP信号通路报告载体； tdTomatoN端融合了一个DD标签； DD标签是一个大小为12KD的FKBP (L106P)去稳定结构域，带有DD标签的目的蛋白将很快被降解； 向培养基中加入Shield1配体后，细胞中目的蛋白的量则迅速提高。。 |
| 产品目录号: | 631087 |
| 稳定性: | 稳表达 或 瞬表达 |
| 组成型/诱导型: | 组成型 |
| 病毒/非病毒: | 非病毒 |
买家导航
pCRE-DD-tdTomato载体质粒基本信息
pCRE-DD-tdTomato质粒图谱载体图谱和pCRE-DD-tdTomato载体序列质粒序列多克隆位点信息
pCRE-DD-tdTomato质粒载体简介
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质粒序列载体序列
LOCUS pCRE-DD-tdTomato 5607 bp DNA circular 21-DEC-2009
COMMENT Created by Clontech Laboratories Inc. http://www.clontech.com
COMMENT Cat. No. 631087,631088
FEATURES Location/Qualifiers
misc_feature 54..140
/label=cAMP\response\element\(CRE)
promoter 147..295
/label=TATA-like\Promoter
CDS 3343..4137
/label=Kan(R)/Neo(R)
rep_origin complement(2363..2818)
/label=f1\origin
rep_origin 3159..3297
/label=SV40\origin
rep_origin 4722..5365
/label=pUC\origin
CDS 353..676
/label=DD
CDS 683..2110
/label=tdTomato
misc_feature 2111..2113
/label=Stop\codon
misc_signal 346..356
/label=Kozak\sequence
polyA_signal 2266..2300
/label=SV40\early\polyA\signals
polyA_signal 4373..4391
/label=HSV\TK\polyA\signals
BASE COUNT 1333 a 1560 c 1530 g 1184 t
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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