pmCherry-1載體描述 pmCherry-1 is a reporter vector that allows the functional analysis of promoters and promoter/enhancer combinations cloned into its multiple cloning site (MCS). The vector encodes mCherry, a mutant fluorescent protein derived from the tetrameric Discosoma sp. red fluorescent protein, DsRed (excitation and emission maxima: 587 nm and 610 nm, respectively; 1). The mCherry coding sequence has been human codon-optimized to allow optimal expression after the insertion of a functional promoter into the MCS (2).
SV40 polyadenylation signals downstream of the mCherry gene direct proper processing of the 3' end of the mCherry 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 (PSV40 e), the
neomycin/kanamycin resistance gene of Tn5, and polyadenylation signals from the herpes simplex virus thymidine kinase (HSV TK) gene. A bacterial promoter (PKanr) upstream of the cassette allows kanamycin resistance in E. coli.
pmCherry can be used as an in vivo reporter of gene expression. Promoters should be cloned into the pmCherry-1 MCS upstream from the mCherry coding sequence. Without the addition of a functionalpromoter, this vector will not express mCherry. The mCherry vector can be transfected into mammalian cells using any standard method. If required, stable transfectants can be selected using G418 (3).
For Western analysis, either the Living Colors DsRed Polyclonal Antibody (Cat. No. 632496) or the DsRed Monoclonal Antibody (Cat. Nos. 632392 and 632393) can be used to detect the mCherry protein. pmCherry-1載體特征 MCS (multiple cloning site): 12–83
mCherry (human codon optimized)
Kozak consensus translation initiation site: 90–100
Start codon (ATG): 97–99; Stop codon: 805-807
SV40 early polyA+ signals
Polyadenylation signals: 959–964 & 988–993; mRNA 3' ends: 997 & 1009
f1 origin of replication: 1056–1511 (complementary)
PKanr (bacterial promoter for Kanr gene expression)
–35 region: 1573–1578; –10 region: 1596–1601
Transcription start point: 1608
PSV40 e (SV40 early promoter and enhancer sequences)
Enhancer (72-bp tandem repeats): 1683–1756 & 1757–1828
21-bp repeats: 1832–1852, 1853–1873 & 1875–1895
Early promoter element: 1918–1924
Major transcription start points: 1904, 1942, 1948 & 1953
SV40 origin of replication: 1852–1987 Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 2036–2038; stop codon: 2828–2830
G→A mutation to remove PstI site: 2218
C→A (Arg→Ser) mutation to remove BssHII site: 2564
HSV TK polyA+ (herpes simplex virus thymidine kinase polyadenylation signals): 3066–3071 & 3079–3084
pUC plasmid replication origin: 3415–4058 質粒的擴增 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) in E. coli hosts.
E. coli replication origin: pUC
Copy number: ~500
Excitation and emission maxima of mCherry
Excitation maximum = 587 nm
Emission maximum = 610 nm
