The vector sequence has been compiled using the information from sequence databases, published literature, and other sources, together with partial sequences obtained by Evrogen. This vector has not been completely sequenced.
pHyPer-nuc is a mammalian expression vector encoding nuclear-targeted HyPer (see reporter description). HyPer codon usage is optimized for high expression in mammalian cells (humanized) [Haas et al., 1996]. Three copies of the nuclear localization signal (NLS) fused to the HyPer C-terminus provide for efficient translocation of HyPer to the nuclei of mammalian cells [Fischer-Fantuzzi and Vesco, 1988].
Ratiometric imaging of HyPer response to H2O2 in HeLa cells nuclei.
HeLa cells expressing HyPer-nuc were plated to glass bottom dishes and exposed to 180 μM H2O2. Images were acquired by Leica AF 6000 LX with 0.5 Hz frequency by sequential illumination of cells via CFP/YFP (excitation/emission) and YFP/YFP filters. Resulting images were obtained by dividing of YFP/YFP images to CFP/YFP images followed by pseudo coloring.
pHyPer-nuc vector can be used as a source of HyPer-NLS hybrid sequence. The vector backbone contains unique restriction sites that permit its excision and further insertion into expression vector of choice. Alternatively, HyPer-NLS coding sequence can be amplified by PCR.
Note: The plasmid DNA was isolated from dam+-methylated
The vector backbone contains immediate early promoter of cytomegalovirus (PCMV IE) for protein expression, SV40 origin for replication in mammalian cells expressing SV40 T-antigen, pUC origin of replication for propagation in
SV40 early promoter (PSV40) provides neomycin resistance gene (Neor) expression to select stably transfected eukaryotic cells using G418. Bacterial promoter (P) provides kanamycin resistance gene expression (Kanr) in
Expression in mammalian cells
pHyPer-nuc vector can be transfected into mammalian cells by any known transfection method. CMV promoter provides strong, constitutive HyPer expression in eukaryotic cells. If required, stable transformants can be selected using G418 [Gorman, 1985].
Suitable host strains for propagation in
Location of features
PCMV IE: 1-589
Enhancer region: 59-465
TATA box: 554-560
Transcription start point: 583
Start codon (ATG): 603-605
HyPer coding sequence: 603-2036
Nuclear localization signals (NLS): 2058-2130
Stop codon: 2148-2150
SV40 early mRNA polyadenylation signal
Polyadenylation signals: 2290-2295 & 2319-2324
mRNA 3' ends: 2328 & 2340
f1 single-strand DNA origin: 2387-2842
Bacterial promoter for expression of Kanr gene
-35 region: 2904-2909
-10 region: 2927-2932
Transcription start point: 2939
SV40 origin of replication: 3183-3318
SV40 early promoter
Enhancer (72-bp tandem repeats): 3016-3087 & 3088-3159
21-bp repeats: 3163-3183, 3184-3204 & 3206-3226
Early promoter element: 3239-3245
Major transcription start points: 3235, 3273, 3279 & 3284
Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 3367-3369
Stop codon: 4159-4161
G->A mutation to remove Pst I site: 3549
C->A (Arg to Ser) mutation to remove BssH II site: 3895
Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 4397-4402 & 4410-4415
pUC plasmid replication origin: 4746-5389
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