pDsRed-Express-C1載體描述 pDsRed-Express-C1 is a mammalian expression vector that encodes DsRed-Express, a variant of Discosoma sp. red fluorescent protein (DsRed; 1). DsRed-Express contains nine amino acid substitutions, which improve the solubility of the protein and reduce the time from transfection to detection of red fluorescence (2). In addition, these substitutions reduce the level of residual green emission (2). When DsRed-Express is expressed in mammalian cell cultures, redemitting cells can be detected by either fluorescence microscopy or flow cytometry 8–12 hours after transfection (DsRed-Express excitation and emission maxima = 557 nm and 579 nm, respectively). Although DsRed-Express most likely forms the same tetrameric structure as wild-type DsRed, DsRed- Express displays a reduced tendency to aggregate (2). The DsRed-Express coding sequence is human codon-optimized for high expression in mammalian cells (3). The multiple cloning site (MCS) in pDsRed-Express-C1 is positioned between the DsRed-Express coding sequence and the SV40 polyadenylation signal (SV40 poly A). Genes cloned into the MCS will be expressed as fusions to the C-terminus of DsRed-Express if they are in the same reading frame as DsRed-Express and there are no intervening stop codons. A Kozak consensus translation initiation site upstream of DsRed-Express increases the translation efficiency in eukaryotic cells (4). SV40 poly A signals downstream of the MCS direct proper processing of the 3' end of mRNA transcripts. The vector backbone also 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)—consisting 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—allows stably transfected eukaryotic cells to be selected using G418. A bacterial promoter upstream of this cassette expresses kanamycin resistance in E. coli.
pDsRed-Express-C1 can be used to construct fusions to the C-terminus of DsRed-Express. If a fusion construct retains the fluorescent properties of the native DsRed-Express protein, its expression can be monitored by flow cytometry and its localization in vivo can be determined by fluorescence microscopy. The target gene should be cloned into pDsRed-Express-C1 so that it is in frame with the DsRed-Express coding sequences, with no intervening in-frame stop codons. The recombinant DsRed-Express vector can be transfected into mammalian cells using any standard transfection method. If required, stable transformants can be selected using G418 (5). pDsRed-Express-C1 can also be used as a cotransfection marker; the unmodified vector will express DsRed-Express. pDsRed-Express-C1載體含有以下元件：
Human cytomegalovirus (CMV) immediate early promoter: 1–589
Enhancer region: 59–465;
TATA box: 554–560
Transcription start point: 583
C→G mutation to remove Sac I site: 569
Discosoma sp. human codon-optimized Red Fluorescent Protein (DsRed-Express) gene
Kozak consensus translation initiation site: 606–616
Start codon (A
TG): 613–615; Stop codon: 1357–1359
CGC→GCC (Arg-2 to Ala) mutation: 616–618
AAG→GAG (Lys-5 to Glu) mutation: 625–627
AAC→GAC (Asn-6 to Asp) mutation: 628–630
ACC→TCC (Thr-21 to Ser) mutation: 673–675
CAC→ACC (His-41 to Thr) mutation: 733–735
AAC→CAG (Asn-42 to Gln) mutation: 736–738
GTG→GCC (Val-44 to Ala) mutation: 742–744
TGC→TCC (Cys-117 to Ser) mutation: 961–963
ACC→GCC (Thr-217 to Ala) mutation: 1261–1263
Last amino acid in DsRed-Express: 1285–1287
MCS: 1288–1344
SV40 early mRNA polyadenylation signal
Polyadenylation signals: 1498–1503 & 1527–1532; mRNA
3' ends: 1536 & 1648
f1 single-strand DNA origin: 1595–2050 (Packages the noncoding strand of DsRed-Express.)
Bacterial promoter for expression of Kanr gene
–35 region: 21
12–2117; –10 region: 2135–2140
Transcription start point: 2147
SV40 origin of replication: 2391–2526
SV40 early promoter
Enhancer (72-bp tandem repeats): 2224–2295 & 2296–2367
21-bp repeats: 2371–2991, 2392–2412 & 2414–2431
Early promoter element: 2447–2453
Major transcription start points: 2443, 2481, 2487 & 2492 Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 2576–2578; stop codon: 3368–3370
G→A mutation to remove Pst I site: 2758
C→A (Arg to Ser) mutation to remove BssH II site: 3104
Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 3606–3611 & 3619–3624
pUC plasmid replication origin: 3955–4598 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
