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.
|Nhe I||Bgl II||Sac I||Hind III||EcoR I||Sal I||Kpn I||Apa I||BamH I||Age I||TagCFP|
|Afe I||Xho I||Pst I||Sac II*|
* – not unique site.
pTagCFP-N is a mammalian expression vector encoding cyan fluorescent protein TagCFP (see reporter description). The vector allows generation of fusions to the TagCFP N-terminus and expression of TagCFP fusions or TagCFP alone in eukaryotic (mammalian) cells.
TagCFP codon usage is optimized for high expression in mammalian cells (humanized) [Haas et al., 1996]. To increase mRNA translation efficiency, Kozak consensus translation initiation site is generated upstream of the TagCFP coding sequence [Kozak, 1987]. Multiple cloning site (MCS) is located between PCMV IE and TagCFP coding sequence.
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
Generation of TagCFP fusion proteins
A localization signal or a gene of interest can be cloned into MCS of the vector. It will be expressed as a fusion to the TagCFP N-terminus when inserted in the same reading frame as TagCFP and no in-frame stop codons are present. The inserted sequence should contain an initiating ATG codon. TagCFP-tagged fusions retain fluorescent properties of the native protein allowing fusion localization in vivo. Unmodified vector will express TagCFP when transfected into eukaryotic (mammalian) cells.
Note: The plasmid DNA was isolated from dam+-methylated
Expression in mammalian cells
pTagCFP-N vector can be transfected into mammalian cells by any known transfection method. CMV promoter provides strong, constitutive expression of TagCFP or its fusions 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: 672-682
Start codon (ATG): 679-681
Stop codon: 1393-1395
SV40 early mRNA polyadenylation signal
Polyadenylation signals: 1549-1554 & 1578-1583
mRNA 3' ends: 1587 & 1599
f1 single-strand DNA origin: 1646-2101
Bacterial promoter for expression of Kanr gene
-35 region: 2163-2168
-10 region: 2186-2191
Transcription start point: 2198
SV40 origin of replication: 2442-2577
SV40 early promoter
Enhancer (72-bp tandem repeats): 2275-2346 & 2347-2418
21-bp repeats: 2422-2442, 2443-2463 & 2465-2485
Early promoter element: 2498-2504
Major transcription start points: 2494, 2532, 2538 & 2543
Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 2626-2628
Stop codon: 3418-3420
G->A mutation to remove Pst I site: 2808
C->A (Arg to Ser) mutation to remove BssH II site: 3154
Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 3656-3661 & 3669-3674
pUC plasmid replication origin: 4005-4648
TagCFP-related materials (also referred to as "Products") are intended for research use only. The Products are covered by European Pat. 06809023 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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