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首页    载体及质粒    pTimer
pTimervectormap

pTimer

pTimer

pTimer

 



编号

载体名称

北京华越洋VECT75353

pTimer

 

pTimer载体基本信息:

载体名称:

pTimer

质粒类型:

无启动子载体;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:

无启动子

载体大小:

4110   bp

5'   测序引物及序列:

DsRed1-N   Sequencing Primer (#6482-1)

3'   测序引物及序列:

--

载体标签:

DsRed1-E5

载体抗性:

卡那霉素

筛选标记:

Neomycin_1.html'   target='_blank'>新霉素(Neomycin

克隆菌株:

DH5α

宿主细胞(系):

真核细胞

备注:

pTimer载体不含启动子,将待研究的目的启动子插入MCS区,用于研究启动子或其它顺式转录元件在细胞内的作用;
  pTimer
含有DsRed1-E5荧光报告基因,在表达初始阶段表现为绿色荧光,随着时间的推移,转变为红色荧光;
 
起到细胞时钟的作用。

稳定性:

瞬表达 稳表达

组成型/诱导型:

--

病毒/非病毒:

非病毒

 

pTimer载体质粒图谱和多克隆位点信息:

 

 

 

pTimer载体描述:

 

pTimer is a prokaryotic expression vector that encodes DsRed1-E5, a fluorescent protein that changes color as it ages. Shortly after translation, DsRed1-E5 emits green light; its red fluorophore emerges later, hours after translation. Because of its predictable color shift, DsRed1-E5 acts as a timer, useful for monitoring changes in gene activity in living cells.

DsRed1-E5 is a mutant of the red fluorescent protein, DsRed1 (1). The cDNA for the wild-type protein, DsRed, was originally isolated by Matz et al., who refer to the protein as drFP583 (2). DsRed1-E5 contains two amino acid substitutions (V105A and S197T), which increase its fluorescence intensity and endow it with a distinct spectral property: As the protein ages, it changes color (3). When first synthesized, DsRed1-E5 is bright green (excitation & emission maxima = 483 nm & 500 nm). As time passes, the green fluorophore undergoes additional changes that cause its fluorescence to shift to longer wavelengthswhen fully matured, the protein is bright red (excitation & emission maxima = 558 nm & 583 nm). In mammalian cells transfected with a Tet-inducible DsRed1-E5 expression vector, the green-to-red transition starts about 3 hours after the protein first becomes fluorescent (3). In addition to the amino acid replacements, DsRed1-E5s coding sequence contains a series of silent base-pair changes, which correspond to human codon-usage preferences, for high expression in mammalian cells (4).

pTimer is primarily intended to serve as a convenient source of the DsRed1-E5 cDNA. The DsRed1-E5 coding sequence is flanked by separate and distinct multiple cloning sites at the 5' and 3' ends so that the gene can easily be excised for use in other expression systems. Alternatively, the DsRed1-E5 coding sequence can be amplified by PCR.

The DsRed1-E5 gene was inserted in frame with the lacZ initiation codon from pUC19 so that DsRed1-E5 is expressed from the lac promoter (Plac) in E. coli host cells. A Kozak consensus sequence is located immediately upstream of DsRed1-E5 to enhance translational efficiency should you wish to express the gene in eukaryotic systems (5). The entire DsRed1-E5 expression cassette in pTimer is supported by a pUC backbone, which contains a high-copy number origin of replication and an ampicillin resistance gene for propagation and selection in E. coli.

When expressed in multicellular organisms, DsRed1-E5s invariable green-to-red shift can be used as a timer to track the on-off phases of gene expression during embryogenesis and cell differentiation (3).

 

Recommended Primer Locations: 472492 and 884907 (DsRed1-C Sequencing Primer, Cat No. 632388, can be used at 884907)

Propagation in E. coli Recommended host strain: DH5α Selectable marker: plasmid confers resistance to ampicillin (50 μg/ml) to E. coli hosts. E. coli replication origin: pUC Copy number: high

 

 

pTimer载体序列:

 

ORIGIN
    1 TAGTTATTAC TAGCGCTACC GGACTCAGAT CTCGAGCTCA AGCTTCGAAT TCTGCAGTCG
   61 ACGGTACCGC GGGCCCGGGA TCCACCGGTC GCCACCATGG TGCGCTCCTC CAAGAACGTC
  121 ATCAAGGAGT TCATGCGCTT CAAGGTGCGC ATGGAGGGCA CCGTGAACGG CCACGAGTTC
  181 GAGATCGAGG GCGAGGGCGA GGGCCGCCCC TACGAGGGCC ACAACACCGT GAAGCTGAAG
  241 GTGACCAAGG GCGGCCCCCT GCCCTTCGCC TGGGACATCC TGTCCCCCCA GTTCCAGTAC
  301 GGCTCCAAGG TGTACGTGAA GCACCCCGCC GACATCCCCG ACTACAAGAA GCTGTCCTTC
  361 CCCGAGGGCT TCAAGTGGGA GCGCGTGATG AACTTCGAGG ACGGCGGCGT GGCGACCGTG
  421 ACCCAGGACT CCTCCCTGCA GGACGGCTGC TTCATCTACA AGGTGAAGTT CATCGGCGTG
  481 AACTTCCCCT CCGACGGCCC CGTGATGCAG AAGAAGACCA TGGGCTGGGA GGCCTCCACC
  541 GAGCGCCTGT ACCCCCGCGA CGGCGTGCTG AAGGGCGAGA TCCACAAGGC CCTGAAGCTG
  601 AAGGACGGCG GCCACTACCT GGTGGAGTTC AAGTCCATCT ACATGGCCAA GAAGCCCGTG
  661 CAGCTGCCCG GCTACTACTA CGTGGACACC AAGCTGGACA TCACCTCCCA CAACGAGGAC
  721 TACACCATCG TGGAGCAGTA CGAGCGCACC GAGGGCCGCC ACCACCTGTT CCTGTAGCGG
  781 CCGCGACTCT AGATCATAAT CAGCCATACC ACATTTGTAG AGGTTTTACT TGCTTTAAAA
  841 AACCTCCCAC ACCTCCCCCT GAACCTGAAA CATAAAATGA ATGCAATTGT TGTTGTTAAC
  901 TTGTTTATTG CAGCTTATAA TGGTTACAAA TAAAGCAATA GCATCACAAA TTTCACAAAT
  961 AAAGCATTTT TTTCACTGCA TTCTAGTTGT GGTTTGTCCA AACTCATCAA TGTATCTTAA
 1021 GGCGTAAATT GTAAGCGTTA ATATTTTGTT AAAATTCGCG TTAAATTTTT GTTAAATCAG
 1081 CTCATTTTTT AACCAATAGG CCGAAATCGG CAAAATCCCT TATAAATCAA AAGAATAGAC
 1141 CGAGATAGGG TTGAGTGTTG TTCCAGTTTG GAACAAGAGT CCACTATTAA AGAACGTGGA
 1201 CTCCAACGTC AAAGGGCGAA AAACCGTCTA TCAGGGCGAT GGCCCACTAC GTGAACCATC
 1261 ACCCTAATCA AGTTTTTTGG GGTCGAGGTG CCGTAAAGCA CTAAATCGGA ACCCTAAAGG
 1321 GAGCCCCCGA TTTAGAGCTT GACGGGGAAA GCCGGCGAAC GTGGCGAGAA AGGAAGGGAA
 1381 GAAAGCGAAA GGAGCGGGCG CTAGGGCGCT GGCAAGTGTA GCGGTCACGC TGCGCGTAAC
 1441 CACCACACCC GCCGCGCTTA ATGCGCCGCT ACAGGGCGCG TCAGGTGGCA CTTTTCGGGG
 1501 AAATGTGCGC GGAACCCCTA TTTGTTTATT TTTCTAAATA CATTCAAATA TGTATCCGCT
 1561 CATGAGACAA TAACCCTGAT AAATGCTTCA ATAATATTGA AAAAGGAAGA GTCCTGAGGC
 1621 GGAAAGAACC AGCTGTGGAA TGTGTGTCAG TTAGGGTGTG GAAAGTCCCC AGGCTCCCCA
 1681 GCAGGCAGAA GTATGCAAAG CATGCATCTC AATTAGTCAG CAACCAGGTG TGGAAAGTCC
 1741 CCAGGCTCCC CAGCAGGCAG AAGTATGCAA AGCATGCATC TCAATTAGTC AGCAACCATA
 1801 GTCCCGCCCC TAACTCCGCC CATCCCGCCC CTAACTCCGC CCAGTTCCGC CCATTCTCCG
 1861 CCCCATGGCT GACTAATTTT TTTTATTTAT GCAGAGGCCG AGGCCGCCTC GGCCTCTGAG
 1921 CTATTCCAGA AGTAGTGAGG AGGCTTTTTT GGAGGCCTAG GCTTTTGCAA AGATCGATCA
 1981 AGAGACAGGA TGAGGATCGT TTCGCATGAT TGAACAAGAT GGATTGCACG CAGGTTCTCC
 2041 GGCCGCTTGG GTGGAGAGGC TATTCGGCTA TGACTGGGCA CAACAGACAA TCGGCTGCTC
 2101 TGATGCCGCC GTGTTCCGGC TGTCAGCGCA GGGGCGCCCG GTTCTTTTTG TCAAGACCGA
 2161 CCTGTCCGGT GCCCTGAATG AACTGCAAGA CGAGGCAGCG CGGCTATCGT GGCTGGCCAC
 2221 GACGGGCGTT CCTTGCGCAG CTGTGCTCGA CGTTGTCACT GAAGCGGGAA GGGACTGGCT
 2281 GCTATTGGGC GAAGTGCCGG GGCAGGATCT CCTGTCATCT CACCTTGCTC CTGCCGAGAA
 2341 AGTATCCATC ATGGCTGATG CAATGCGGCG GCTGCATACG CTTGATCCGG CTACCTGCCC
 2401 ATTCGACCAC CAAGCGAAAC ATCGCATCGA GCGAGCACGT ACTCGGATGG AAGCCGGTCT
 2461 TGTCGATCAG GATGATCTGG ACGAAGAGCA TCAGGGGCTC GCGCCAGCCG AACTGTTCGC
 2521 CAGGCTCAAG GCGAGCATGC CCGACGGCGA GGATCTCGTC GTGACCCATG GCGATGCCTG
 2581 CTTGCCGAAT ATCATGGTGG AAAATGGCCG CTTTTCTGGA TTCATCGACT GTGGCCGGCT
 2641 GGGTGTGGCG GACCGCTATC AGGACATAGC GTTGGCTACC CGTGATATTG CTGAAGAGCT
 2701 TGGCGGCGAA TGGGCTGACC GCTTCCTCGT GCTTTACGGT ATCGCCGCTC CCGATTCGCA
 2761 GCGCATCGCC TTCTATCGCC TTCTTGACGA GTTCTTCTGA GCGGGACTCT GGGGTTCGAA
 2821 ATGACCGACC AAGCGACGCC CAACCTGCCA TCACGAGATT TCGATTCCAC CGCCGCCTTC
 2881 TATGAAAGGT TGGGCTTCGG AATCGTTTTC CGGGACGCCG GCTGGATGAT CCTCCAGCGC
 2941 GGGGATCTCA TGCTGGAGTT CTTCGCCCAC CCTAGGGGGA GGCTAACTGA AACACGGAAG
 3001 GAGACAATAC CGGAAGGAAC CCGCGCTATG ACGGCAATAA AAAGACAGAA TAAAACGCAC
 3061 GGTGTTGGGT CGTTTGTTCA TAAACGCGGG GTTCGGTCCC AGGGCTGGCA CTCTGTCGAT
 3121 ACCCCACCGA GACCCCATTG GGGCCAATAC GCCCGCGTTT CTTCCTTTTC CCCACCCCAC
 3181 CCCCCAAGTT CGGGTGAAGG CCCAGGGCTC GCAGCCAACG TCGGGGCGGC AGGCCCTGCC
 3241 ATAGCCTCAG GTTACTCATA TATACTTTAG ATTGATTTAA AACTTCATTT TTAATTTAAA
 3301 AGGATCTAGG TGAAGATCCT TTTTGATAAT CTCATGACCA AAATCCCTTA ACGTGAGTTT
 3361 TCGTTCCACT GAGCGTCAGA CCCCGTAGAA AAGATCAAAG GATCTTCTTG AGATCCTTTT
 3421 TTTCTGCGCG TAATCTGCTG CTTGCAAACA AAAAAACCAC CGCTACCAGC GGTGGTTTGT
 3481 TTGCCGGATC AAGAGCTACC AACTCTTTTT CCGAAGGTAA CTGGCTTCAG CAGAGCGCAG
 3541 ATACCAAATA CTGTCCTTCT AGTGTAGCCG TAGTTAGGCC ACCACTTCAA GAACTCTGTA
 3601 GCACCGCCTA CATACCTCGC TCTGCTAATC CTGTTACCAG TGGCTGCTGC CAGTGGCGAT
 3661 AAGTCGTGTC TTACCGGGTT GGACTCAAGA CGATAGTTAC CGGATAAGGC GCAGCGGTCG
 3721 GGCTGAACGG GGGGTTCGTG CACACAGCCC AGCTTGGAGC GAACGACCTA CACCGAACTG
 3781 AGATACCTAC AGCGTGAGCT ATGAGAAAGC GCCACGCTTC CCGAAGGGAG AAAGGCGGAC
 3841 AGGTATCCGG TAAGCGGCAG GGTCGGAACA GGAGAGCGCA CGAGGGAGCT TCCAGGGGGA
 3901 AACGCCTGGT ATCTTTATAG TCCTGTCGGG TTTCGCCACC TCTGACTTGA GCGTCGATTT
 3961 TTGTGATGCT CGTCAGGGGG GCGGAGCCTA TGGAAAAACG CCAGCAACGC GGCCTTTTTA
 4021 CGGTTCCTGG CCTTTTGCTG GCCTTTTGCT CACATGTTCT TTCCTGCGTT ATCCCCTGAT
 4081 TCTGTGGATA ACCGTATTAC CGCCATGCAT
// 

 

pTimer载体其他相关的其他类型载体:

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pHY300PLK

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pHY300

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pHT304

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pHT43

pXMJ19

pHT01

pVLT33

pHSE-Luc

pUG6

pHIS1525

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pHGF9050

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pHD_v2

pTA-Luc

pHcRed1-DR

pTALETF_v2 (NN)

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pTALETF_v2 (NI)

pHCMC05

pTALETF_v2 (NG)

pHCMC04

pTALETF_v2 (HD)

pGRE-luc

pTALEN_v2 (NN)

pGFP315

pTALEN_v2 (NI)

pGFP22

pTALEN_v2 (NG)

pGEM-3

pTALEN_v2 (HD)

pGAS-TA-Luc

pSRE-Luc

pE2F-TA-Luc

pSH65

pDsRed-Express-DR

pSH63

pDsRed-Express2-1

pSH47

pDsRed-Express-1

pSG1154

pDsRed2-1

pset152

pDG1363

pRK2013

pCSN44

pQE-TriSystem

pDD-tdTomato Reporter

pOS7001

pCRE-Luc

pNZ8048

pCR-Blunt

pNN_v2

pCMV-Raf-1

pNI v2

pCMV-p53

pNG_v2

pCMV-IκBα

pMUTIN4

pCMV-CREB

pMG36e

pCaspase3-sensor

pMetLuc2-Reporter

pCantab 5E

pMetLuc2-Control

pBT2

pmCherry-1

pBR322

pMA09-J

pBIG2R-HPH2

pMA09-H

pAP-1-Luc

pMA5MCS

pAN8-1

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pkc1139

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HRV 3C protease

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GG499

pAN7-1

p53-Luc

pAP1(PMA)-TA-Luc