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.
|Nhe I||Bgl II||Sac I||Hind III||EcoR I||Sal I||Kpn I||Apa I||BamH I||KillerRed|
|Afe I||Xho I||Pst I||Sac II||Age I||Nco I*|
* – not unique site.
pKillerRed-N is a mammalian expression vector encoding photosensitizer KillerRed (see reporter description). The vector allows generation of fusions to the KillerRed N-terminus and expression of KillerRed fusions or KillerRed alone in eukaryotic (mammalian) cells.
KillerRed codon usage is optimized for high expression in mammalian cells (humanized) [Haas et al., 1996]. To increase mRNA translation efficiency, Kozak consensus translation initiation site is generated upstream of the KillerRed coding sequence [Kozak, 1987]. Multiple cloning site (MCS) is located between PCMV IE and KillerRed coding sequence.
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
Generation of KillerRed fusion proteins
A localization signal or a gene of interest can be cloned into MCS of the vector. It will be expressed as a fusion to the KillerRed N-terminus when inserted in the same reading frame as KillerRed and no in-frame stop codons are present. The inserted sequence should contain an initiating ATG codon. KillerRed-tagged fusions retain fluorescent properties of the native protein allowing fusion localization in vivo. Unmodified vector will express KillerRed when transfected into eukaryotic (mammalian) cells.
Note: The plasmid DNA was isolated from dam+-methylated
Expression in mammalian cells
pKillerRed-N vector can be transfected into mammalian cells by any known transfection method. CMV promoter provides strong, constitutive expression of KillerRed or its fusions in eukaryotic cells. If required, stable transformants can be selected using G418 [Gorman, 1985].
Note: KillerRed shows no cell toxic effects before light activation. Upon green light irradiation KillerRed generates reactive oxygen species (ROS) that damage the neighboring molecules.
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
Kozak consensus translation initiation site: 672-682
Start codon (ATG): 679-681
Stop codon: 1396-1398
SV40 early mRNA polyadenylation signal
Polyadenylation signals: 1552-1557 & 1581-1586
mRNA 3' ends: 1590 & 1602
f1 single-strand DNA origin: 1649-2104
Bacterial promoter for expression of Kanr gene
-35 region: 2166-2171
-10 region: 2189-2194
Transcription start point: 2201
SV40 origin of replication: 2445-2580
SV40 early promoter
Enhancer (72-bp tandem repeats): 2278-2349 & 2350-2421
21-bp repeats: 2425-2445, 2446-2466 & 2468-2488
Early promoter element: 2501-2507
Major transcription start points: 2497, 2535, 2541 & 2546
Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 2629-2631
Stop codon: 3421-3423
G->A mutation to remove Pst I site: 2811
C->A (Arg to Ser) mutation to remove BssH II site: 3157
Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 3659-3664 & 3672-3677
pUC plasmid replication origin: 4008-4651
KillerRed-related materials (also referred to as "Products") are intended for research use only.
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