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-paxillin is a mammalian expression vector encoding mKate2-paxillin fusion protein (see reporter description). The vector can be used for fluorescent labeling of paxillin in living cells.
N-terminal mKate2 fusion construct with chicken paxillin in human cervical adenocarcinoma cells (HeLa).
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]. Chicken paxillin is fused to the mKate2 N-terminus.
pmKate2-paxillin vector can be used as a source of mKate2-paxillin 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-paxillin vector can be transfected into mammalian cells by any known transfection method. CMV promoter provides strong, constitutive expression of the mKate2-paxillin 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
Paxillin-mKate2 fusion: 600-3038
Start codon (ATG): 600-602
Last amino acid in Paxillin: 2274-2276
Stop codon: 3039-3041
SV40 early mRNA polyadenylation signal
Polyadenylation signals: 3194-3199 & 3223-3228
mRNA 3' ends: 3232 & 3244
f1 single-strand DNA origin: 3291-3746
Bacterial promoter for expression of Kanr gene
-35 region: 3808-3813
-10 region: 3831-3836
Transcription start point: 3843
SV40 origin of replication: 4087-4222
SV40 early promoter
Enhancer (72-bp tandem repeats): 3920-3991 & 3992-4063
21-bp repeats: 4067-4087, 4088-4108 & 4110-4130
Early promoter element: 4143-4149
Major transcription start points: 4139, 4177, 4183 & 4188
Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 4271-4273
Stop codon: 5063-5065
G->A mutation to remove Pst I site: 4453
C->A (Arg to Ser) mutation to remove BssH II site: 4799
Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 5301-5306 & 5314-5319
pUC plasmid replication origin: 5650-6293
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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