pDsRed2-1載體描述 pDsRed2-1 encodes DsRed2, a DsRed variant engineered for faster maturation and lower non-specific aggregation. Derived from the Discosoma sp. red fluorescent protein (drFP583; 1), DsRed2, like its progenitor DsRed1, contains a series of silent base-pair changes that correspond to human codon-usage preferences for high expression in mammalian cells (2). In addition to these changes, DsRed2 contains six amino acid substitutions: V105A, I161T, and S197A, which result in the more rapid appearance of red fluorescence in transfected cell lines; and R2A, K5E, and K9T, which prevent the protein from aggregating. (DsRed2 may, however, form the same tetrameric structure as DsRed1 [3].) In mammalian cell cultures when DsRed2 is expressed constitutively, red-emitting cells can be detected by fluorescence microscopy within 24 hours of transfection. Large insoluble aggregates of protein, often observed in bacterial and mammalian cell systems expressing DsRed1, are dramatically reduced in cells expressing DsRed2. The faster-maturing, more soluble red fluorescent protein is also well tolerated by host cells; mammalian cell cultures transfected with DsRed2 show no obvious signs of reduced viability—in those cell lines tested, cells expressing DsRed2 display the same morphology (e.g., adherence, light-refraction) and growth characteristics as non-transfected controls.
pDsRed2-1 is a promoterless DsRed2 vector that can be used to monitor transcription from different promoters and promoter/enhancer combinations inserted into the multiple cloning site (MCS). Sequences upstream of DsRed2 have been converted to a Kozak consensus translation initiation site (4) to increase translation efficiency in eukaryotic cells. SV40 polyadenylation signals downstream of the DsRed2 gene direct proper processing of the 3' end of the DsRed2 mRNA. The vector backbone contains an SV40 origin for replication in mammalian cells expressing the SV40 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.
DsRed2 can be used as an in vivo reporter of gene expression. Promoters should be cloned into the pDsRed2-1 MCS upstream from the DsRed2 coding sequence. Without addition of a functional promoter, this vector will not express DsRed2. The recombinant DsRed2 vector can be transfected into mammalian cells using any standard transfection method. If required, stable transfectants can be selected using G418 (5). pDsRed2-1載體含有以下元件： MCS: 12–83
Discosoma sp. human codon-optimized Red Fluorescent Protein (DsRed2) gene
Kozak consensus translation initiation site: 90–100
Start codon (ATG): 97–99; Stop codon: 772–774
SV40 early mRNA polyadenylation signal
Polyadenylation signals: 926–931 & 955–960
mRNA 3' ends: 964 & 976
f1 single-strand DNA origin: 1023–1478
(Packages noncoding strand of DsRed2.)
Ampicillin resistance (b-lactamase) promoter
–35 region: 1540–1545; –10 region: 1563–1568
Transcription start point: 1575
SV40 origin of replication: 1819–1954
SV40 early promoter
Enhancer (72-bp tandem repeats): 1650–1723 & 1724–1795
21-bp repeats: 1799–1819, 1820–1840 & 1842–1862
Early promoter element: 1875–1881
Major transcription start points: 1871, 1909, 1915 & 1920 Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 2003–2005; stop codon: 2795–2797
G→A mutation to remove Pst I site: 2185
C→A (Arg→Ser) mutation to remove BssH II site: 2531
Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 3033–3038 & 3046–3051
pUC plasmid replication origin: 3382–4025
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
Red Fluorescent Protein (DsRed2)
Excitation/Emission Maxima: 558 nm / 583 nm
