pDsRed-Express-N1載體描述 pDsRed-Express-N1 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 (listed on page 2), 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-N1 is positioned between the immediate early promoter of CMV (PCMV IE) and the DsRed-Express coding sequence. Genes cloned into the MCS are expressed as fusions to the N-terminus of DsRed-Express if they are in the same reading frame as DsRed-Express and there are no intervening stop codons. Sequences upstream of DsRed-Express have been converted to a Kozak consensus translation initiation site to increase translation efficiency in eukaryotic cells (4). SV40 polyadenylation signals downstream of the DsRed-Express gene direct proper processing of the 3' end of the DsRed-Express 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 confers kanamycin resistance to E. coli.
pDsRed-Express-N1 can be used to construct fusions to the N-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-N1 so that it is in frame with the DsRed-Express coding sequence, with no intervening in-frame stop codons. The inserted gene should include an initiating ATG codon. Recombinant pDsRed-Express-N1 can be transfected into mammalian cells using any standard transfection method. If required, stable transfectants can be selected using G418 (5). pDsRed-Express-N1 can also be used as a cotransfection marker; the unmodified vector will express DsRed-Express. pDsRed-Express-N1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
MCS: 591–671
Discosoma sp. Red Fluorescent Protein (DsRed-Express) gene
Kozak consensus translation initiation site: 672–682
Start codon (ATG): 679–681; Stop codon: 1354–1356
CGC→GCC (Arg-2 to Ala) mutation: 682–684
AAG→GAG (Lys-5 to Glu) mutation: 691–693
AAC→GAC (Asn-6 to Asp) mutation: 694–696
ACC→TCC (Thr-21 to Ser) mutation: 739–741
CAC→ACC (His-41 to Thr) mutation: 799–801
AAC→CAG (Asn-42 to Gln) mutation: 802–804
GTG→GCC (Val-44 to Ala) mutation: 808–810
TGC→TCC (Cys-117 to Ser) mutation: 1027–1029
ACC→GCC (Thr-217 to Ala) mutation: 1327–1329
SV40 early mRNA polyadenylation signal
Polyadenylation signals: 1508–1513 & 1537–1542; mRNA 3' ends: 1546 & 1558
f1 single-strand DNA origin: 1605–2060 (Packages the noncoding strand of DsRed-Express.)
Bacterial promoter for expression of Kanr gene:
–35 region: 2122–2127; –10 region: 2145–2150
Transcription start point: 2157
SV40 origin of replication: 2401–2536
SV40 early promoter
Enhancer (72-bp tandem repeats): 2234–2305 & 2306–2377
21-bp repeats: 2381–2401, 2402–2422 & 2424–2444
Early promoter element: 2457–2463
Major transcription start points: 2453, 2491, 2497 & 2502 Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences: Start codon (ATG): 2585–2587; Stop codon: 3377–3379
G→A mutation to remove Pst I site: 2767
C→A (Arg to Ser) mutation to remove BssH II site: 3113
Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 3615–3620 & 3628–3633
pUC plasmid replication origin: 3964–4607
Sequencing primer locations
DsRed1-N Sequencing Primer (Cat. No. 632387; 5'-GTACTGGAACTGGGGGGACAG-3'): 879–859
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 the JM109 or XL1-Blue strains..
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/ColE1
Excitation and emission maxima of DsRed-Express
Excitation maximum = 557 nm
Emission maximum = 579 nm
