pCRE-DD-tdTomato

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载体基本信息

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: Kanamycin.html' target='_blank'>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

载体质粒图谱和多克隆位点信息

pCRE-DD-tdTomato载体图谱



pCRE-DD-tdTomato 载体特征

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.

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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