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.
pmKate2-endo is a mammalian expression vector intended for far-red fluorescent labeling of vesicles of the endocytic pathway [Adamson et al., 1992], allowing the monitoring of intracellular membrane traffic during endocytosis in living cells. The vector encodes far-red fluorescent protein mKate2 (see reporter description) targeted to endosomes by human RhoB GTPase fused to the mKate2 C-terminus. The fusion also contains c-Myc epitope tag.
HeLa cell expressing fusion of human RhoB GTPase and c-Myc epitope tag with mKate2.
The fusion protein is localized in the endosomes.
mKate2 codon usage is optimized for high expression in mammalian cells (humanized) [Haas et al., 1996].
pmKate2-endo vector can be used as a source of mKate2-RhoB hybrid sequence. The vector backbone contains unique restriction sites that permit its excision and further insertion into expression vector of choice.
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
pmKate2-endo vector can be transfected into mammalian cells by any known transfection method. CMV promoter provides strong, constitutive expression of the mKate2-RhoB fusion 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
mKate2-RhoB GTPase fusion
Start codon (ATG): 613-615
Start of mKate2 coding sequence (ATG): 613-615
Last amino acid in mKate2: 1312-1314
c-Myc epitope: 1357-1359
RhoB GTPase: 1383-1968
Stop codon: 1969-1971
SV40 early mRNA polyadenylation signal
Polyadenylation signals: 2163-2168 & 2192-2197
mRNA 3' ends: 2201 & 2213
f1 single-strand DNA origin: 2260-2715
Bacterial promoter for expression of Kanr gene
-35 region: 2777-2782
-10 region: 2800-2805
Transcription start point: 2812
SV40 origin of replication: 3056-3191
SV40 early promoter
Enhancer (72-bp tandem repeats): 2889-2960 & 2961-3032
21-bp repeats: 3036-3056, 3057-3077 & 3079-3099
Early promoter element: 3112-3118
Major transcription start points: 3108, 3146, 3152 & 3157
Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 3240-3242
Stop codon: 4032-4034
G->A mutation to remove Pst I site: 3422
C->A (Arg to Ser) mutation to remove BssH II site: 3768
Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 4270-4275 & 4283-4288
pUC plasmid replication origin: 4619-5262
mKate2-related materials (also referred to as "Products") are intended for research use only. The Products are covered by U.S. Pat. 7,638,615; European Pat. 1994149; and other 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.
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