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-keratin is a mammalian expression vector encoding mKate2-keratin fusion protein (see reporter description). The vector can be used for fluorescent labeling of cytokeratin-18 in living cells.
Transiently transfected HeLa cells expressing mKate2 fusion with cytokeratin-18.
Scale bar represents 10 μm. Image from Shcherbo et al., 2009.
mKate2 codon usage is optimized for high expression in mammalian cells (humanized) [Haas et al., 1996]. Human cytokeratin-18 is fused to the mKate2 N-terminus.
pmKate2-keratin vector can be used as a source of mKate2-keratin 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-keratin vector can be transfected into mammalian cells by any known transfection method. CMV promoter provides strong, constitutive expression of the mKate2-keratin 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
Keratin-mKate2 fusion: 600-2636
Start codon (ATG): 600-602
Last amino acid in Keratin: 1887-1889
Stop codon: 2637-2639
SV40 early mRNA polyadenylation signal
Polyadenylation signals: 2792-2797 & 2821-2826
mRNA 3' ends: 2830 & 2842
f1 single-strand DNA origin: 2889-3344
Bacterial promoter for expression of Kanr gene
-35 region: 3406-3411
-10 region: 3429-3434
Transcription start point: 3441
SV40 origin of replication: 3685-3820
SV40 early promoter
Enhancer (72-bp tandem repeats): 3518-3589 & 3590-3661
21-bp repeats: 3665-3685, 3686-3706 & 3708-3728
Early promoter element: 3741-3747
Major transcription start points: 3737, 3775, 3781 & 3786
Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 3869-3871
Stop codon: 4661-4663
G->A mutation to remove Pst I site: 4051
C->A (Arg to Ser) mutation to remove BssH II site: 4397
Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 4899-4904 & 4912-4917
pUC plasmid replication origin: 5248-5891
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.
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