pTurboFP635-N

pTurboFP635-N vector

cat.# FP722

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.


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vector information:
ProductCat.#SizePrice
pTurboFP635-NFP72220 μg€ 320 / 160*
*50% discount on the second and subsequent vectors encoding same fluorescent protein or sensor ordered by the same customer.
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Vector typemammalian expression vector
ReporterTurboFP635
Reporter codon usagemammalian
Promoter for TurboFP635PCMV IE
Host cellsmammalian
Selectionprokaryotic – kanamycin
eukaryotic – neomycin (G418)
Replicationprokaryotic – pUC ori
eukaryotic – SV40 ori
Use TurboFP635 expression in mammalian cells; generation of fusions to the TurboFP635 N-terminus
Multiple cloning site (MCS)
Nhe I Bgl II Sac I Hind III EcoR I Sal I Kpn I Apa I BamH I Age I TurboFP635
Afe I Xho I Pst I Sac II Sma I/Xma I Nco I*
GCT A.GC G.CT A.CCG.GAC.TC A.GAT. CT C. GAG. CTC. AAG.CTT. C GA.ATT. C TG.CA G. TCG.AC G.GTA. CC G.C GG. G CC.C G G.G AT.CC A.CCG.GT C.GCC.A CC. ATG.G ...

* – not unique site.

Vector description

pTurboFP635-N is a mammalian expression vector encoding far-red fluorescent protein TurboFP635 (see reporter description). The vector allows generation of fusions to the TurboFP635 N-terminus and expression of TurboFP635 fusions or TurboFP635 alone in eukaryotic (mammalian) cells.

TurboFP635 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 TurboFP635 coding sequence [Kozak, 1987]. Multiple cloning site (MCS) is located between PCMV IE and TurboFP635 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 E. coli, and f1 origin for single-stranded DNA production. SV40 polyadenylation signals (SV40 poly A) direct proper processing of the 3'-end of the reporter mRNA.

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 E. coli. Kanr/Neor gene is linked with herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signals.


Generation of TurboFP635 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 TurboFP635 N-terminus when inserted in the same reading frame as TurboFP635 and no in-frame stop codons are present. The inserted sequence should contain an initiating ATG codon. TurboFP635-tagged fusions retain fluorescent properties of the native protein allowing fusion localization in vivo. Unmodified vector will express TurboFP635 when transfected into eukaryotic (mammalian) cells.

Note: The plasmid DNA was isolated from dam+-methylated E. coli. Therefore some restriction sites are blocked by methylation. If you wish to digest the vector using such sites you will need to transform the vector into a dam- host and make fresh DNA.


Expression in mammalian cells

pTurboFP635-N vector can be transfected into mammalian cells by any known transfection method. CMV promoter provides strong, constitutive expression of TurboFP635 or its fusions in eukaryotic cells. If required, stable transformants can be selected using G418 [Gorman, 1985].


Propagation in E. coli

Suitable host strains for propagation in E. coli include DH5alpha, HB101, XL1-Blue, and other general purpose strains. Plasmid incompatibility group is pMB1/ColE1. The vector confers resistance to kanamycin (30 μg/ml) to E. coli hosts. Copy number in E. coli is about 500.


Location of features

PCMV IE: 1-589
Enhancer region: 59-465
TATA box: 554-560
Transcription start point: 583
MCS: 591-671
TurboFP635
Kozak consensus translation initiation site: 672-682
Start codon (ATG): 679-681
Stop codon: 1384-1386
SV40 early mRNA polyadenylation signal
Polyadenylation signals: 1540-1545 & 1569-1574
mRNA 3' ends: 1578 & 1590
f1 single-strand DNA origin: 1637-2092
Bacterial promoter for expression of Kanr gene
-35 region: 2154-2159
-10 region: 2177-2182
Transcription start point: 2189
SV40 origin of replication: 2433-2568
SV40 early promoter
Enhancer (72-bp tandem repeats): 2266-2337 & 2338-2409
21-bp repeats: 2413-2433, 2434-2454 & 2456-2476
Early promoter element: 2489-2495
Major transcription start points: 2485, 2523, 2529 & 2534
Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 2617-2619
Stop codon: 3409-3411
G->A mutation to remove Pst I site: 2799
C->A (Arg to Ser) mutation to remove BssH II site: 3145
Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 3647-3652 & 3660-3665
pUC plasmid replication origin: 3996-4639


References:

  • Gorman C. High efficiency gene transfer into mammalian cells. In DNA cloning: A Practical Approach, Vol. II. Ed. D. M. Glover. (IRL Press, Oxford, U.K.). 1985; 143-90.
  • Haas J, Park EC, Seed B. Codon usage limitation in the expression of HIV-1 envelope glycoprotein. Curr Biol. 1996; 6 (3):315-24. / pmid: 8805248
  • Kozak M. An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 1987; 15 (20):8125-48. / pmid: 3313277

Notice to Purchaser:

TurboFP635-related materials (also referred to as "Products") are intended for research use only. The Products are covered by U.S. Pat. 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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