pDsRed-Express-DR載體描述 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載體含有以下元件： 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 gene
Neomycin 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
