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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. |
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Vector description
pKillerRed-mem is a mammalian expression vector encoding membrane-targeted KillerRed (see protein description). KillerRed localized on cellular membrane can be used for effective light-induced cell killing.
Note: Comparing to the mitochondrially targeted KillerRed, irradiation of membrane-localized KillerRed leads to even more effective and fast cell death (within 10-30 min). Moreover, membrane-targeted KillerRed was shown to be suitable for the light induced cell killing within a developing zebrafish.
KillerRed codon usage is optimized for high expression in mammalian cells (humanized) [Haas et al., 1996]. Membrane localization signal (MLS) of neuromodulin is linked to the KillerRed N-terminus. The MLS (N-terminal 20 amino acid residues of neuromodulin) contains a signal for posttranslational palmitoylation of cysteines 3 and 4 that targets KillerRed to cellular membranes [Skene and Virag, 1989].
pKillerRed-mem can be used as a source of MLS-KillerRed hybrid sequence. The vector backbone contains unique restriction sites that permit it excision and further insertion into expression vector of choice.
Note: The plasmid DNA was isolated from dam+-methylated E.coli. Therefore some restriction sites are blocked by methylation. If you wish to digest the vector using such sites you will need to transform the vector into a dam- host and make fresh DNA.
The vector backbone also contains immediate early promoter of cytomegalovirus (PCMV IE) for reporter expression, SV40 origin for replication in mammalian cells expressing SV40 T-antigen, pUC origin of replication for propagation in E. coli and f1 origin for single-stranded DNA production. SV40 polyadenylation signals (SV40 poly A) direct proper processing of the 3'-end of the reporter mRNA.
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 E. coli. Kanr/Neor gene is linked with herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signals.
Expression in mammalian cells
The vector can be transfected into mammalian cells by any known transfection method. CMV promoter provides strong, constitutive expression of memrane-targeted KillerRed in many cell types. 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.
Propagation in E. coli
Suitable host strains for propagation in E. coli include DH5alpha, HB101, XL1-Blue, and other general purpose strains. Plasmid incompatibility group is pMB1/ColE1. The vector confers resistance to kanamycin (30 μg/ml) to E. coli hosts. Copy number in E. coli is about 500.
Location of features
PCMV IE: 1-589
Enhancer region: 59-465
TATA box: 554-560
Transcription start point: 583
KillerRed-mem fusion
Start codon (ATG): 679-681
Neuromodulin N-terminal sequence (mem): 679-738
Start of KillerRed coding sequence (ATG): 739-741
Stop codon: 1450-1452
SV40 early mRNA polyadenylation signal
Polyadenylation signals: 1606-1611 & 1635-1640
mRNA 3' ends: 1644 & 1656
f1 single-strand DNA origin: 1703-2158
Eukaryotic promoter for expression of Kanr gene
-35 region: 2220-2225
-10 region: 2243-2248
Transcription start point: 2255
SV40 origin of replication: 2499-2634
SV40 early promoter
Enhancer (72-bp tandem repeats): 2332-2403 & 2404-2475
21-bp repeats: 2479-2499, 2500-2520 & 2522-2542
Early promoter element: 2555-2561
Major transcription start points: 2551, 2589, 2595 & 2600
Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 2683-2685
Stop codon: 3475-3477
G->A mutation to remove Pst I site: 2865
C->A (Arg to Ser) mutation to remove BssH II site: 3211
Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 3713-3718 & 3726-3731
pUC plasmid replication origin: 4062-4705
References:
- Gorman C. High efficiency gene transfer into mammalian cells. In DNA cloning: A Practical Approach, Vol. II. Ed. D. M. Glover. (IRL Press, Oxford, U.K.). 1985; 143–90.
- Haas J, Park EC, Seed B. Codon usage limitation in the expression of HIV-1 envelope glycoprotein. Curr Biol. 1996; 6 (3):315-24. / pmid: 8805248
- Skene JH, Virag I. Posttranslational membrane attachment and dynamic fatty acylation of a neuronal growth cone protein, GAP-43. J Cell Biol. 1989; 108 (2):613-24. / pmid: 2918027
Notice to Purchaser:
Evrogen FP-related products are intended for research use only and covered by Evrogen Patents and/or Patent applications pending. By use of these products, you accept the terms and conditions of the applicable Limited Use Label License.
The CMV promoter is covered under U.S. Patents 5,168,062 and 5,385,839, and its use is permitted for research purposes only. Any other use of the CMV promoter requires a license from the University of Iowa Research Foundation, 214 Technology Innovation Center, Iowa City, IA 52242.
MATERIAL SAFETY DATA SHEET INFORMATION
To the best of our knowledge, these products do not require a Material Safety Data Sheet. However, all the properties of these products (and, if applicable, each of their components) have not been thoroughly investigated. Therefore, we recommend that you use gloves and eye protection, and wear a laboratory coat when working with these products.
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