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.
pFusionRed-MAP4 is a mammalian expression vector encoding FusionRed-MAP4 fusion protein (see reporter description). The vector can be used for fluorescent labeling of microtubules in living cells.
Human bone osteosarcoma cell expressing fusion of FusionRed with human microtubule-associated protein MAP4.
Movie from Shemiakina et al., 2012.
FusionRed codon usage is optimized for high expression in mammalian cells (humanized) [Haas et al., 1996]. Microtubule binding domain of mouse microtubule-associated protein 4 (MAP4) is fused to the FusionRed C-terminus. To increase mRNA translation efficiency, Kozak consensus translation initiation site is generated upstream of the FusionRed-MAP4 coding sequence [Kozak, 1987].
pFusionRed-MAP4 vector can be used as a source of FusionRed-MAP4 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
pFusionRed-MAP4 vector can be transfected into mammalian cells by any known transfection method. CMV promoter provides strong, constitutive expression of the FusionRed-MAP4 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
Kozak consensus translation initiation site: 606-616
Start codon (ATG): 613-615
Last amino acid in FusionRed: 1306-1308
Microtubule binding domain of MAP4: 1351-2571
Stop codon: 2569-2571
SV40 early mRNA polyadenylation signal
Polyadenylation signals: 2732-2737 & 2761-2766
mRNA 3' ends: 2770 & 2782
f1 single-strand DNA origin: 2829-3284
Bacterial promoter for expression of Kanr gene
-35 region: 3346-3351
-10 region: 3369-3374
Transcription start point: 3381
SV40 origin of replication: 3625-3760
SV40 early promoter
Enhancer (72-bp tandem repeats): 3458-3529 & 3530-3601
21-bp repeats: 3605-3625, 3626-3646 & 3648-3668
Early promoter element: 3681-3687
Major transcription start points: 3677, 3715, 3721 & 3726
Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 3809-3811
Stop codon: 4601-4603
G->A mutation to remove Pst I site: 3991
C->A (Arg to Ser) mutation to remove BssH II site: 4337
Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 4839-4844 & 4852-4857
pUC plasmid replication origin: 5188-5831
FusionRed-related materials (also referred to as "Products") are intended for research use only. The Products are covered by 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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