pZsGreen1-DR载体描述
pZsGreen1-DR is a promoterless vector that encodes ZsGreen1-DR, a destabilized variant of
Zoanthus sp. green fluorescent protein (ZsGreen; 1). The ZsGreen1 coding sequence contains
a series of silent base-pair changes, which correspond to human codon-usage preferences, for
optimal expression in mammalian cells (2). In contrast to the original protein, ZsGreen1-DR has a
short half-life, making it well suited for studies that require rapid reporter turnover. This destabilized
variant 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 short-lived proteins in mammalian cells (3).
This region of MODC contains a PEST sequence that targets the protein for degradation, resulting
in rapid protein turnover (3,4). Three point mutations in this sequence further reduce the protein
half-life (3). Sequences upstream of ZsGreen1-DR have been converted to a Kozak consensus
translation initiation site (5) to enhance translation efficiency in eukaryotic cells. A single amino acid
substitution (Asn-65 to Met) has been made to enhance the emission characteristics of ZsGreen1
(excitation maximum = 496 nm; emission maximum = 506 nm).
pZsGreen1-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 ZsGreen1-DR
coding sequence. Downstream SV40 polyadenylation signals direct proper processing of the 3'
end of the ZsGreen1-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.
使用：
ZsGreen1-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 ZsGreen1.
Promoter/enhancer elements should be inserted in the MCS upstream of the ZsGreen1-DR coding sequence. Without the addition of a
functional promoter, this vector will not express ZsGreen1-DR. The recombinant pZsGreen1-DR vector can be transfected into mammalian
cells using any standard transfection method. If required, stable transformants can be selected using G418 (6).
Location of features
MCS: 12–83
Destabilized Zoanthus sp. Green Fluorescent Protein (ZsGreen1-DR) gene
Kozak consensus translation initiation site: 83–93
Start codon (ATG): 90–92; Stop codon: 915–917
Asn-65 to Met mutation (A→T, C→G ): 286, 287
Mouse ornithine decarboxylase PEST sequence: 795–917
SV40 early mRNA polyadenylation signal
Polyadenylation signals: 1070–1075 & 1099–1104
mRNA 3' ends: 1108 & 1120
f1 single-strand DNA origin: 1167–1622
(Packages noncoding strand of ZsGreen1-DR.)
Ampicillin resistance (β-lactamase) promoter
–35 region: 1684–1689; –10 region: 1707–1712
Transcription start point: 1719
SV40 origin of replication: 1963–2098
SV40 early promoter
Enhancer (72-bp tandem repeats): 1794–1867 & 1868–1939
21-bp repeats: 1943–1963, 1964–1984 & 1986–2006
Early promoter element: 2019–2025
Major transcription start points: 2015, 2053, 2059 & 2064 Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 2147–2149; stop codon: 2939–2941
G→A mutation to remove Pst I site: 2329
C→A (Arg→Ser) mutation to remove BssH II site: 2675
Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 3177–3182 & 3190–3195
pUC plasmid replication origin: 3526–4169
Excitation and emission maxima of ZsGreen1
Excitation maximum = 496 nm
Emission maximum = 506 nm
