pTimer

pTimer

pTimer载体基本信息：

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 wavelengths—when 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-E5’s 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-E5’s 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: 472–492 and 884–907 (DsRed1-C Sequencing Primer, Cat No. 632388, can be used at 884–907)

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：