pDsRed-Express2

pDsRed-Express2

pDsRed-Express2载体基本信息

pDsRed-Express2载体质粒图谱和多克隆位点信息

pDsRed-Express2载体描述 

pDsRed-Express2 is a prokaryotic expression vector that encodes DsRed-Express2, a variant of the Discosoma sp. red fluorescent protein, DsRed (1). DsRed-Express2 retains the fast maturation and high photostability characteristic of its predecessor, DsRed-Express (2), and has been engineered (through additional amino acid substitutions) for increased solubility and reduced cytotoxicity (3). Although it most likely forms the same tetrameric structure as wild-type DsRed, DsRed-Express2 displays a greatly reduced tendency to aggregate; this results in minimal cytotoxicity, which makes DsRed-Express2 much better suited for in vivo applications involving sensitive cells, such as primary or stem cells. DsRed-Express2 also exhibits extremely low residual green fluorescence, which allows cells expressing the protein to be effectively separated from other fluorescently labeled cell populations by flow cytometry.

In pDsRed-Express2, the DsRed-Express2 coding sequence is flanked by separate and distinct multiple cloning sites (i.e., the 5' MCS and 3' MCS) that make it easy to excise the gene for use in other cloning applications. In E. coli, DsRed-Express2 is expressed from the lac promoter (Plac) as a fusion with several amino acids, including the first five amino acids of the LacZ protein. Note, however, that if the DsRed-Express2 coding sequence is excised using a restriction site in the 5' MCS, the protein will no longer be expressed as a fusion (as it is when it is expressed from the lac promoter). A Kozak consensus sequence is located immediately upstream of the DsRed-Express2 coding sequence to enhance translational efficiency in eukaryotic cells (4). The entire DsRed-Express2 expression cassette in pDsRed-Express2 is supported by a pUC19 backbone, which contains a high-copy number origin of replication and an ampicillin resistance gene (Ampr) for propagation and selection in E. coli.

pDsRed-Express2 is primarily intended to serve as a source of DsRed-Express2 cDNA. The flanking MCS regions make it possible to excise the DsRed-Express2 coding sequence and insert it into other vector systems. The vector can also be used to express DsRed-Express2 in bacteria. Cells expressing DsRed-Express2 (excitation and emission maxima: 554 nm and 591 nm, respectively) can be detected by either fluorescence microscopy or flow cytometry 8–12 hours after transfection.

For Western analysis, DsRed-Express2 can be detected with either the Living Colors DsRed Polyclonal Anti-body or the Living Colors DsRed Monoclonal Antibody.

pDsRed-Express2载体含有以下元件： 

Plac (lac Promoter): 95–178

CAP binding site: 111–124

–35 region: 143–148; –10 region: 167–172

lac operator: 179–199

 lacZ-DsRed-Express2 fusion expressed in E. coli

Ribosome binding site: 206–209

Start codon (ATG): 217–219; Stop codon 961–963

 5' MCS (5' multiple cloning site): 234–292

 DsRed-Express2 (Discosoma sp. red fluorescent protein variant)

Kozak consensus translation initiation site: 282–292

Start codon (ATG): 289–291; Stop codon: 964–966

 3' MCS (3' multiple cloning site): 966–1065

 Ampr (Ampicillin resistance gene): 1511–2371

 pUC origin of replication: 2519–3161

Propagation in E. coli 

Recommended host strain: DH5α

 Selectable marker: plasmid confers resistance to ampicillin (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 DsRed-Express2

 Excitation maximum = 554 nm

 Emission maximum = 591 nm

载体序列

ORIGIN

    1 AGCGCCCAAT ACGCAAACCG CCTCTCCCCG CGCGTTGGCC GATTCATTAA TGCAGCTGGC

   61 ACGACAGGTT TCCCGACTGG AAAGCGGGCA GTGAGCGCAA CGCAATTAAT GTGAGTTAGC

  121 TCACTCATTA GGCACCCCAG GCTTTACACT TTATGCTTCC GGCTCGTATG TTGTGTGGAA

  181 TTGTGAGCGG ATAACAATTT CACACAGGAA ACAGCTATGA CCATGATTAC GCCAAGCTTG

  241 CATGCCTGCA GGTCGACTCT AGAGGATCCC CGGGTACCGG TCGCCACCAT GGATAGCACT

  301 GAGAACGTCA TCAAGCCCTT CATGCGCTTC AAGGTGCACA TGGAGGGCTC CGTGAACGGC

  361 CACGAGTTCG AGATCGAGGG CGAGGGCGAG GGCAAGCCCT ACGAGGGCAC CCAGACCGCC

  421 AAGCTGCAGG TGACCAAGGG CGGCCCCCTG CCCTTCGCCT GGGACATCCT GTCCCCCCAG

  481 TTCCAGTACG GCTCCAAGGT GTACGTGAAG CACCCCGCCG ACATCCCCGA CTACAAGAAG

  541 CTGTCCTTCC CCGAGGGCTT CAAGTGGGAG CGCGTGATGA ACTTCGAGGA CGGCGGCGTG

  601 GTGACCGTGA CCCAGGACTC CTCCCTGCAG GACGGCACCT TCATCTACCA CGTGAAGTTC

  661 ATCGGCGTGA ACTTCCCCTC CGACGGCCCC GTAATGCAGA AGAAGACTCT GGGCTGGGAG

  721 CCCTCCACCG AGCGCCTGTA CCCCCGCGAC GGCGTGCTGA AGGGCGAGAT CCACAAGGCG

  781 CTGAAGCTGA AGGGCGGCGG CCACTACCTG GTGGAGTTCA AGTCAATCTA CATGGCCAAG

  841 AAGCCCGTGA AGCTGCCCGG CTACTACTAC GTGGACTCCA AGCTGGACAT CACCTCCCAC

  901 AACGAGGACT ACACCGTGGT GGAGCAGTAC GAGCGCGCCG AGGCCCGCCA CCACCTGTTC

  961 CAGTAGCGGC CGCGACTCTA GAATTCCAAC TGAGCGCCGG TCGCTACCAT TACCAACTTG

 1021 TCTGGTGTCA AAAATAATAG GCCTACTAGT CGGCCGTACG GGCCCTTTCG TCTCGCGCGT

 1081 TTCGGTGATG ACGGTGAAAA CCTCTGACAC ATGCAGCTCC CGGAGACGGT CACAGCTTGT

 1141 CTGTAAGCGG ATGCCGGGAG CAGACAAGCC CGTCAGGGCG CGTCAGCGGG TGTTGGCGGG

 1201 TGTCGGGGCT GGCTTAACTA TGCGGCATCA GAGCAGATTG TACTGAGAGT GCACCATATG

 1261 CGGTGTGAAA TACCGCACAG ATGCGTAAGG AGAAAATACC GCATCAGGCG GCCTTAAGGG

 1321 CCTCGTGATA CGCCTATTTT TATAGGTTAA TGTCATGATA ATAATGGTTT CTTAGACGTC

 1381 AGGTGGCACT TTTCGGGGAA ATGTGCGCGG AACCCCTATT TGTTTATTTT TCTAAATACA

 1441 TTCAAATATG TATCCGCTCA TGAGACAATA ACCCTGATAA ATGCTTCAAT AATATTGAAA

 1501 AAGGAAGAGT ATGAGTATTC AACATTTCCG TGTCGCCCTT ATTCCCTTTT TTGCGGCATT

 1561 TTGCCTTCCT GTTTTTGCTC ACCCAGAAAC GCTGGTGAAA GTAAAAGATG CTGAAGATCA

 1621 GTTGGGTGCA CGAGTGGGTT ACATCGAACT GGATCTCAAC AGCGGTAAGA TCCTTGAGAG

 1681 TTTTCGCCCC GAAGAACGTT TTCCAATGAT GAGCACTTTT AAAGTTCTGC TATGTGGCGC

 1741 GGTATTATCC CGTATTGACG CCGGGCAAGA GCAACTCGGT CGCCGCATAC ACTATTCTCA

 1801 GAATGACTTG GTTGAGTACT CACCAGTCAC AGAAAAGCAT CTTACGGATG GCATGACAGT

 1861 AAGAGAATTA TGCAGTGCTG CCATAACCAT GAGTGATAAC ACTGCGGCCA ACTTACTTCT

 1921 GACAACGATC GGAGGACCGA AGGAGCTAAC CGCTTTTTTG CACAACATGG GGGATCATGT

 1981 AACTCGCCTT GATCGTTGGG AACCGGAGCT GAATGAAGCC ATACCAAACG ACGAGCGTGA

 2041 CACCACGATG CCTGTAGCAA TGGCAACAAC GTTGCGCAAA CTATTAACTG GCGAACTACT

 2101 TACTCTAGCT TCCCGGCAAC AATTAATAGA CTGGATGGAG GCGGATAAAG TTGCAGGACC

 2161 ACTTCTGCGC TCGGCCCTTC CGGCTGGCTG GTTTATTGCT GATAAATCTG GAGCCGGTGA

 2221 GCGTGGGTCT CGCGGTATCA TTGCAGCACT GGGGCCAGAT GGTAAGCCCT CCCGTATCGT

 2281 AGTTATCTAC ACGACGGGGA GTCAGGCAAC TATGGATGAA CGAAATAGAC AGATCGCTGA

 2341 GATAGGTGCC TCACTGATTA AGCATTGGTA ACTGTCAGAC CAAGTTTACT CATATATACT

 2401 TTAGATTGAT TTAAAACTTC ATTTTTAATT TAAAAGGATC TAGGTGAAGA TCCTTTTTGA

 2461 TAATCTCATG ACCAAAATCC CTTAACGTGA GTTTTCGTTC CACTGAGCGT CAGACCCCGT

 2521 AGAAAAGATC AAAGGATCTT CTTGAGATCC TTTTTTTCTG CGCGTAATCT GCTGCTTGCA

 2581 AACAAAAAAA CCACCGCTAC CAGCGGTGGT TTGTTTGCCG GATCAAGAGC TACCAACTCT

 2641 TTTTCCGAAG GTAACTGGCT TCAGCAGAGC GCAGATACCA AATACTGTTC TTCTAGTGTA

 2701 GCCGTAGTTA GGCCACCACT TCAAGAACTC TGTAGCACCG CCTACATACC TCGCTCTGCT

 2761 AATCCTGTTA CCAGTGGCTG CTGCCAGTGG CGATAAGTCG TGTCTTACCG GGTTGGACTC

 2821 AAGACGATAG TTACCGGATA AGGCGCAGCG GTCGGGCTGA ACGGGGGGTT CGTGCACACA

 2881 GCCCAGCTTG GAGCGAACGA CCTACACCGA ACTGAGATAC CTACAGCGTG AGCTATGAGA

 2941 AAGCGCCACG CTTCCCGAAG GGAGAAAGGC GGACAGGTAT CCGGTAAGCG GCAGGGTCGG

 3001 AACAGGAGAG CGCACGAGGG AGCTTCCAGG GGGAAACGCC TGGTATCTTT ATAGTCCTGT

 3061 CGGGTTTCGC CACCTCTGAC TTGAGCGTCG ATTTTTGTGA TGCTCGTCAG GGGGGCGGAG

 3121 CCTATGGAAA AACGCCAGCA ACGCGGCCTT TTTACGGTTC CTGGCCTTTT GCTGGCCTTT

 3181 TGCTCACATG TTCTTTCCTG CGTTATCCCC TGATTCTGTG GATAACCGTA TTACCGCCTT

 3241 TGAGTGAGCT GATACCGCTC GCCGCAGCCG AACGACCGAG CGCAGCGAGT CAGTGAGCGA

 3301 GGAAGCGGAA G

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