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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
pKindling-Red-mito is a mammalian expression vector encoding mitochondria-targeted kindling red fluorescent protein KFP-Red (see protein description). The vector can be used for kindling red fluorescent labeling of mitochondria.
KFP-Red codon usage is optimized for high expression in mammalian cells (humanized) [Haas et al., 1996]. A mitochondrial targeting sequence (MTS) is fused to the KFP-Red N-terminus. MTS was derived from the subunit VIII of human cytochrome C oxidase [Rizzuto et al., 1989; Rizzuto et al., 1995].
pKindling-Red-mito can be used as a source of MTS-KFP-Red 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 mitochondria-targeted KFP-Red in many cell types resulting in kindling red fluorescent labeling of mithochondria. If required, stable transformants can be selected using G418 [Gorman, 1985].
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
KFP-Red-mito fusion
Start codon (ATG): 597-599
Mitochondrial targeting sequence (MTS): 597-683
Start of XGFP coding sequence (ATG): 705-707
Stop codon: 1401-1403
SV40 early mRNA polyadenylation signal
Polyadenylation signals: 1556-1561 & 1585-1590
mRNA 3' ends: 1594 & 1606
f1 single-strand DNA origin: 1653-2108
Eukaryotic promoter for expression of Kanr gene
-35 region: 2170-2175
-10 region: 2193-2198
Transcription start point: 2205
SV40 origin of replication: 2449-2584
SV40 early promoter
Enhancer (72-bp tandem repeats): 2282-2353 & 2354-2425
21-bp repeats: 2429-2449, 2450-2470 & 2472-2492
Early promoter element: 2505-2511
Major transcription start points: 2501, 2539, 2545 & 2550
Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 2633-2635
Stop codon: 3425-3427
G->A mutation to remove Pst I site: 2815
C->A (Arg to Ser) mutation to remove BssH II site: 3161
Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 3663-3668 & 3676-3681
pUC plasmid replication origin: 4012-4655
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
- Rizzuto R, Nakase H, Darras B, Francke U, Fabrizi GM, Mengel T, Walsh F, Kadenbach B, DiMauro S, Schon EA. A gene specifying subunit VIII of human cytochrome c oxidase is localized to chromosome 11 and is expressed in both muscle and non-muscle tissues. J Biol Chem. 1989; 264 (18):10595-600. / pmid: 2543673
- Rizzuto R, Brini M, Pizzo P, Murgia M, Pozzan T. Chimeric green fluorescent protein as a tool for visualizing subcellular organelles in living cells. Curr Biol. 1995; 5 (6):635-42. / pmid: 7552174
Notice to Purchaser:
Photoactivatable FP-related products are intended to be used by academic (non-commercial) entities and for research purposes only. Any use of the proprietary nucleic acid or protein other than for research use or by a commercial entity is strictly prohibited. Transfer of this product by purchaser to any other party is specifically prohibited.
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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