pDsRed-Express-DR

pDsRed-Express-DR

pDsRed-Express-DR载体基本信息：

pDsRed-Express-DR载体质粒图谱和多克隆位点信息

pDsRed-Express-DR载体描述：

pDsRed-Express-DR is a promoterless vector that encodes DsRed-Express-DR, a destabilized variant of Discosoma sp. red fluorescent protein (DsRed). In contrast to the original protein, which is extremely stable, DsRed-Express-DR has a short half-life, making it well suited for studies that require rapid reporter turnover. Though its geneology can be traced to wild-type DsRed (1), DsRed-Express-DR is directly derived from DsRed-Express, a DsRed variant engineered for rapid fluorescence development (2, 3). DsRed-Express contains nine amino acid substitutions (listed on page 2), which improve the protein’s solubility and decrease the time from transfection to detection of red fluorescence (2). The substitutions also reduce the level of residual green emission (2). The destabilized variant, DsRed-Express-DR, was constructed by fusing the C-terminus of the protein to amino acid residues 422–461 of mouse ornithine decarboxylase (MODC), one of the most shortlived proteins in mammalian cells (4). This region of MODC contains a PEST sequence that targets the protein for degradation, resulting in rapid protein turnover (4, 5). The DsRed-Express-DR gene is human codon-optimized for high expression in mammalian cells (6), and sequences upstream of the gene have been converted to a Kozak consensus translation initiation site (7) to enhance translation efficiency in eukaryotic cells.

pDsRed-Express-DR can be used to monitor transcription from different promoters and promoter/ enhancer combinations inserted into the multiple cloning site (MCS), located upstream of the DsRed-Express-DR coding sequence. SV40 polyadenylation signals downstream of the DsRed- Express-DR gene direct proper processing of the 3' end of the DsRed-Express-DR mRNA. 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. This cassette consists of the SV40 early promoter, the neomycin/kanamycin resistance gene of Tn5, and polyadenylation signals from the Herpes simplex virus thymidine kinase (HSV TK) gene. A bacterial promoter upstream of the cassette expresses kanamycin resistance in E. coli.

DsRed-Express-DR can be used as an in vivo reporter of gene expression. Because of its rapid turnover rate, its expression from a promoter of interest provides a more accurate assessment of the promoter's activity over time than does the more stable DsRed-Express. Promoter/enhancer elements should be inserted into the MCS upstream of the DsRed-Express-DR coding sequence. Without the addition of a functional promoter, this vector will not express DsRed-Express-DR. The recombinant pDsRed-Express-DR vector can be transfected into mammalian cells using any standard transfection method. If required, stable transformants can be selected using G418 (8).

pDsRed-Express-DR载体含有以下元件：

 MCS: 12–89

 Destabilized Discosoma sp. Red Fluorescent Protein (DsRed-Express-DR) gene

Kozak consensus translation initiation site: 90–100

Start codon (ATG): 97–99; Stop codon: 904–906

CGC→GCC (Arg-2 to Ala) mutation: 100–102

AAG→GAG (Lys-5 to Glu) mutation: 109–111

AAC→GAC (Asn-6 to Asp) mutation: 112–114

ACC→TCC (Thr-21 to Ser) mutation: 157–159

CAC→ACC (His-41 to Thr) mutation: 217–219

AAC→CAG (Asn-42 to Gln) mutation: 220–222

GTG→GCC (Val-44 to Ala) mutation: 226–228

TGC→TCC (Cys-117 to Ser) mutation: 445–447

ACC→GCC (Thr-217 to Ala) mutation: 745–747

Mouse ornithine decarboxylase PEST sequence: 784–906

 SV40 early mRNA polyadenylation signal

Polyadenylation signals: 1059–1064 & 1088–1093

mRNA 3' ends: 1097 & 1109

 f1 single-strand DNA origin: 1156–1611

(Packages noncoding strand of DsRed-Express-DR.)

 Ampicillin resistance (β-lactamase) promoter

–35 region: 1673–1678; –10 region: 1696–1701

Transcription start point: 1708

 SV40 origin of replication: 1952–2087

 SV40 early promoter

Enhancer (72-bp tandem repeats): 1783–1856 & 1857–1928

21-bp repeats: 1932–1952, 1953–1973 & 1975–1995

Early promoter element: 2008–2014

Major transcription start points: 2004, 2042, 2048 & 2053

 Kanamycin/neomycin resistance geneNeomycin phosphotransferase coding sequences:

Start codon (ATG): 2136–2138; stop codon: 2928–2930

G→A mutation to remove Pst I site: 2318

C→A (Arg→Ser) mutation to remove BssH II site: 2664

 Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal

Polyadenylation signals: 3166–3171 & 3179–3184

 pUC plasmid replication origin: 3515–4158

Propagation in E. Coli

 Suitable 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) to E. coli hosts.

 E. coli replication origin: pUC

 Copy number: ~500

 Plasmid incompatibility group: pMB1/Col E1

Excitation and emission maxima of DsRed-Express

 Excitation maximum = 557 nm

 Emission maximum = 579 nm

pDsRed-Express-DR载体序列：

ORIGIN

    1 TAGTTATTAC TAGCGCTACC GGACTCAGAT CTCGAGCTCA AGCTTCGAAT TCTGCAGTCG

   61 ACGGTACCGC GGGCCCGGGA TCCACCGGTC GCCACCATGG CCTCCTCCGA GGACGTCATC

  121 AAGGAGTTCA TGCGCTTCAA GGTGCGCATG GAGGGCTCCG TGAACGGCCA CGAGTTCGAG

  181 ATCGAGGGCG AGGGCGAGGG CCGCCCCTAC GAGGGCACCC AGACCGCCAA GCTGAAGGTG

  241 ACCAAGGGCG GCCCCCTGCC CTTCGCCTGG GACATCCTGT CCCCCCAGTT CCAGTACGGC

  301 TCCAAGGTGT ACGTGAAGCA CCCCGCCGAC ATCCCCGACT ACAAGAAGCT GTCCTTCCCC

  361 GAGGGCTTCA AGTGGGAGCG CGTGATGAAC TTCGAGGACG GCGGCGTGGT GACCGTGACC

  421 CAGGACTCCT CCCTGCAGGA CGGCTCCTTC ATCTACAAGG TGAAGTTCAT CGGCGTGAAC

  481 TTCCCCTCCG ACGGCCCCGT AATGCAGAAG AAGACTATGG GCTGGGAGGC CTCCACCGAG

  541 CGCCTGTACC CCCGCGACGG CGTGCTGAAG GGCGAGATCC ACAAGGCCCT GAAGCTGAAG

  601 GACGGCGGCC ACTACCTGGT GGAGTTCAAG TCCATCTACA TGGCCAAGAA GCCCGTGCAG

  661 CTGCCCGGCT ACTACTACGT GGACTCCAAG CTGGACATCA CCTCCCACAA CGAGGACTAC

  721 ACCATCGTGG AGCAGTACGA GCGCGCCGAG GGCCGCCACC ACCTGTTCCT GACTAGTGAT

  781 ATCAGCCATG GCTTCCCGCC GGCGGTGGCG GCGCAGGATG ATGGCACGCT GCCCATGTCT

  841 TGTGCCCAGG AGAGCGGGAT GGACCGTCAC CCTGCAGCCT GTGCTTCTGC TAGGATCAAT

  901 GTGTAGGCGG CCGCGACTCT AGATCATAAT CAGCCATACC ACATTTGTAG AGGTTTTACT

  961 TGCTTTAAAA AACCTCCCAC ACCTCCCCCT GAACCTGAAA CATAAAATGA ATGCAATTGT

 1021 TGTTGTTAAC TTGTTTATTG CAGCTTATAA TGGTTACAAA TAAAGCAATA GCATCACAAA

 1081 TTTCACAAAT AAAGCATTTT TTTCACTGCA TTCTAGTTGT GGTTTGTCCA AACTCATCAA

 1141 TGTATCTTAA GGCGTAAATT GTAAGCGTTA ATATTTTGTT AAAATTCGCG TTAAATTTTT

 1201 GTTAAATCAG CTCATTTTTT AACCAATAGG CCGAAATCGG CAAAATCCCT TATAAATCAA

 1261 AAGAATAGAC CGAGATAGGG TTGAGTGTTG TTCCAGTTTG GAACAAGAGT CCACTATTAA

 1321 AGAACGTGGA CTCCAACGTC AAAGGGCGAA AAACCGTCTA TCAGGGCGAT GGCCCACTAC

 1381 GTGAACCATC ACCCTAATCA AGTTTTTTGG GGTCGAGGTG CCGTAAAGCA CTAAATCGGA

 1441 ACCCTAAAGG GAGCCCCCGA TTTAGAGCTT GACGGGGAAA GCCGGCGAAC GTGGCGAGAA

 1501 AGGAAGGGAA GAAAGCGAAA GGAGCGGGCG CTAGGGCGCT GGCAAGTGTA GCGGTCACGC

 1561 TGCGCGTAAC CACCACACCC GCCGCGCTTA ATGCGCCGCT ACAGGGCGCG TCAGGTGGCA

 1621 CTTTTCGGGG AAATGTGCGC GGAACCCCTA TTTGTTTATT TTTCTAAATA CATTCAAATA

 1681 TGTATCCGCT CATGAGACAA TAACCCTGAT AAATGCTTCA ATAATATTGA AAAAGGAAGA

 1741 GTCCTGAGGC GGAAAGAACC AGCTGTGGAA TGTGTGTCAG TTAGGGTGTG GAAAGTCCCC

 1801 AGGCTCCCCA GCAGGCAGAA GTATGCAAAG CATGCATCTC AATTAGTCAG CAACCAGGTG

 1861 TGGAAAGTCC CCAGGCTCCC CAGCAGGCAG AAGTATGCAA AGCATGCATC TCAATTAGTC

 1921 AGCAACCATA GTCCCGCCCC TAACTCCGCC CATCCCGCCC CTAACTCCGC CCAGTTCCGC

 1981 CCATTCTCCG CCCCATGGCT GACTAATTTT TTTTATTTAT GCAGAGGCCG AGGCCGCCTC

 2041 GGCCTCTGAG CTATTCCAGA AGTAGTGAGG AGGCTTTTTT GGAGGCCTAG GCTTTTGCAA

 2101 AGATCGATCA AGAGACAGGA TGAGGATCGT TTCGCATGAT TGAACAAGAT GGATTGCACG

 2161 CAGGTTCTCC GGCCGCTTGG GTGGAGAGGC TATTCGGCTA TGACTGGGCA CAACAGACAA

 2221 TCGGCTGCTC TGATGCCGCC GTGTTCCGGC TGTCAGCGCA GGGGCGCCCG GTTCTTTTTG

 2281 TCAAGACCGA CCTGTCCGGT GCCCTGAATG AACTGCAAGA CGAGGCAGCG CGGCTATCGT

 2341 GGCTGGCCAC GACGGGCGTT CCTTGCGCAG CTGTGCTCGA CGTTGTCACT GAAGCGGGAA

 2401 GGGACTGGCT GCTATTGGGC GAAGTGCCGG GGCAGGATCT CCTGTCATCT CACCTTGCTC

 2461 CTGCCGAGAA AGTATCCATC ATGGCTGATG CAATGCGGCG GCTGCATACG CTTGATCCGG

 2521 CTACCTGCCC ATTCGACCAC CAAGCGAAAC ATCGCATCGA GCGAGCACGT ACTCGGATGG

 2581 AAGCCGGTCT TGTCGATCAG GATGATCTGG ACGAAGAGCA TCAGGGGCTC GCGCCAGCCG

 2641 AACTGTTCGC CAGGCTCAAG GCGAGCATGC CCGACGGCGA GGATCTCGTC GTGACCCATG

 2701 GCGATGCCTG CTTGCCGAAT ATCATGGTGG AAAATGGCCG CTTTTCTGGA TTCATCGACT

 2761 GTGGCCGGCT GGGTGTGGCG GACCGCTATC AGGACATAGC GTTGGCTACC CGTGATATTG

 2821 CTGAAGAGCT TGGCGGCGAA TGGGCTGACC GCTTCCTCGT GCTTTACGGT ATCGCCGCTC

 2881 CCGATTCGCA GCGCATCGCC TTCTATCGCC TTCTTGACGA GTTCTTCTGA GCGGGACTCT

 2941 GGGGTTCGAA ATGACCGACC AAGCGACGCC CAACCTGCCA TCACGAGATT TCGATTCCAC

 3001 CGCCGCCTTC TATGAAAGGT TGGGCTTCGG AATCGTTTTC CGGGACGCCG GCTGGATGAT

 3061 CCTCCAGCGC GGGGATCTCA TGCTGGAGTT CTTCGCCCAC CCTAGGGGGA GGCTAACTGA

 3121 AACACGGAAG GAGACAATAC CGGAAGGAAC CCGCGCTATG ACGGCAATAA AAAGACAGAA

 3181 TAAAACGCAC GGTGTTGGGT CGTTTGTTCA TAAACGCGGG GTTCGGTCCC AGGGCTGGCA

 3241 CTCTGTCGAT ACCCCACCGA GACCCCATTG GGGCCAATAC GCCCGCGTTT CTTCCTTTTC

 3301 CCCACCCCAC CCCCCAAGTT CGGGTGAAGG CCCAGGGCTC GCAGCCAACG TCGGGGCGGC

 3361 AGGCCCTGCC ATAGCCTCAG GTTACTCATA TATACTTTAG ATTGATTTAA AACTTCATTT

 3421 TTAATTTAAA AGGATCTAGG TGAAGATCCT TTTTGATAAT CTCATGACCA AAATCCCTTA

 3481 ACGTGAGTTT TCGTTCCACT GAGCGTCAGA CCCCGTAGAA AAGATCAAAG GATCTTCTTG

 3541 AGATCCTTTT TTTCTGCGCG TAATCTGCTG CTTGCAAACA AAAAAACCAC CGCTACCAGC

 3601 GGTGGTTTGT TTGCCGGATC AAGAGCTACC AACTCTTTTT CCGAAGGTAA CTGGCTTCAG

 3661 CAGAGCGCAG ATACCAAATA CTGTCCTTCT AGTGTAGCCG TAGTTAGGCC ACCACTTCAA

 3721 GAACTCTGTA GCACCGCCTA CATACCTCGC TCTGCTAATC CTGTTACCAG TGGCTGCTGC

 3781 CAGTGGCGAT AAGTCGTGTC TTACCGGGTT GGACTCAAGA CGATAGTTAC CGGATAAGGC

 3841 GCAGCGGTCG GGCTGAACGG GGGGTTCGTG CACACAGCCC AGCTTGGAGC GAACGACCTA

 3901 CACCGAACTG AGATACCTAC AGCGTGAGCT ATGAGAAAGC GCCACGCTTC CCGAAGGGAG

 3961 AAAGGCGGAC AGGTATCCGG TAAGCGGCAG GGTCGGAACA GGAGAGCGCA CGAGGGAGCT

 4021 TCCAGGGGGA AACGCCTGGT ATCTTTATAG TCCTGTCGGG TTTCGCCACC TCTGACTTGA

 4081 GCGTCGATTT TTGTGATGCT CGTCAGGGGG GCGGAGCCTA TGGAAAAACG CCAGCAACGC

 4141 GGCCTTTTTA CGGTTCCTGG CCTTTTGCTG GCCTTTTGCT CACATGTTCT TTCCTGCGTT

 4201 ATCCCCTGAT TCTGTGGATA ACCGTATTAC CGCCATGCAT

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