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||EcoR I*||Sal I||Sac II||Sma I/Xma I||Age I||NirFP|
|Afe I||Xho I||Hind III||Pst I||Kpn I||Apa I||BamH I*||Nco I*|
* – not unique site.
peNirFP-N is a mammalian expression vector encoding near-infrared fluorescent protein NirFP (see reporter description). The vector allows generation of fusions to the NirFP N-terminus and expression of NirFP fusions or NirFP alone in eukaryotic (mammalian) cells.
NirFP 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 NirFP coding sequence [Kozak, 1987]. Fragments of exons 2 and 3 and intron 2 of human beta globin gene are added in the 3’ UTR of NirFP coding sequence in order to increase the protein expression level. Multiple cloning site (MCS) is located between PCMV IE and NirFP 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 NirFP 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 NirFP N-terminus when inserted in the same reading frame as NirFP and no in-frame stop codons are present. The inserted sequence should contain an initiating ATG codon. NirFP-tagged fusions retain fluorescent properties of the native protein allowing fusion localization in vivo. Unmodified vector will express NirFP when transfected into eukaryotic (mammalian) cells.
Note: The plasmid DNA was isolated from dam+-methylated
Expression in mammalian cells
peNirFP-N vector can be transfected into mammalian cells by any known transfection method. CMV promoter provides strong, constitutive expression of NirFP 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: 1381-1383
Fragment of human beta globin (HBB) gene: 1392-2510
Last 35 bp of HBB exon 2: 1392-1426
HBB intron 2: 1427-2277
First 233 bp of HBB exon3: 2278-2510
SV40 early mRNA polyadenylation signal
Polyadenylation signals: 2652-2657 & 2681-2686
mRNA 3' ends: 2690 & 2702
f1 single-strand DNA origin: 2749-3204
Bacterial promoter for expression of Kanr gene
-35 region: 3266-3271
-10 region: 3289-3294
Transcription start point: 3301
SV40 origin of replication: 3545-3680
SV40 early promoter
Enhancer (72-bp tandem repeats): 3378-3449 & 3450-3521
21-bp repeats: 3525-3545, 3546-3566 & 3568-3588
Early promoter element: 3601-3607
Major transcription start points: 3597, 3635, 3641 & 3646
Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 3729-3731
Stop codon: 4521-4523
G->A mutation to remove Pst I site: 3911
C->A (Arg to Ser) mutation to remove BssH II site: 4257
Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 4759-4764 & 4772-4777
pUC plasmid replication origin: 5108-5751
NirFP-related materials (also referred to as "Products") are intended for research use only. The Products are covered by U.S. Pat. 7,972,834; 8,138,320; 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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