Eng | Ru

    Gateway® TurboGFP-N

Gateway® TurboGFP-N entry clone

cat.# FP522

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.


Download
vector information:
ProductCat.#SizePrice
Gateway® TurboGFP-NFP52220 μg€ 320 / 160*
*50% discount on the second and subsequent vectors encoding same fluorescent protein or sensor ordered by the same customer.
The price does not include delivery. The price varies in different countries. Please contact your local distributor for exact prices and delivery information.

Vector typeGateway® entry clone
ReporterTurboGFP
Reporter codon usagemammalian
Promoter for TurboGFPNO
Host cellsprokaryotic
Selectionkanamycin
ReplicationpUC ori
Use Generation of fusions to the N-terminus of TurboGFP; transfer of the construct encoding TurboGFP or its fusion into Gateway® destination vectors
Multiple cloning site (MCS)
attL1 site Bgl II Sac I Hind III EcoR I Sal I Kpn I Apa I* BamH I Age I TurboGFP
Afe I Xho I Pst I* Sac II Sma I/Xma I Nco I*
...AGG.CTG.CT 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 AG.AGC

* – not unique site.

Vector description

Gateway® TurboGFP-N entry clone is a vector containing green fluorescent protein TurboGFP gene variant with codon usage optimized for high expression in mammalian cells (humanized) [Haas et al., 1996] (see reporter description). TurboGFP coding sequence is flanked by attL1 and attL2 sites allowing easy site-specific recombination. The Invitrogen Gateway® Technology provides a rapid and highly efficient way to transfer the TurboGFP gene into a number of Gateway® destination vectors for expression in different experimental systems. Multiple cloning site (MCS) located at the 5'-end of TurboGFP gene allows to generate fusions to the TurboGFP N-terminus for expression, localization and cellular dynamics studies.

To increase mRNA translation efficiency, Kozak consensus translation initiation site is generated upstream of the TurboGFP coding sequence [Kozak, 1987].

The vector backbone contains pUC origin of replication and kanamycin resistance gene (Kanr) for propagation and selection in E. coli.


Generation of TurboGFP fusion proteins

A localization signal or a gene of interest can be cloned into MCS of the vector both before and after site-specific recombination with a destination vector. It will be expressed as a fusion to the TurboGFP N-terminus when inserted in the same reading frame as TurboGFP and no in-frame stop codons are present.

Alternatively, TurboGFP gene can be fused to the 3'-end of a gene of interest by LR recombination of the Gateway® TurboGFP-N with a destination vector containing this gene in a correct reading frame.

TurboGFP-tagged fusions retain fluorescent properties of the native protein allowing fusion localization in vivo.

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.


LR site-specific recombination

Please refer to Invitrogen Gateway® Technology description for detailed instructions regarding LR site-specific recombination reaction. In general, to transfer TurboGFP gene or TurboGFP-fusion construct into the destination vector you will need:

  - Purified plasmid DNA of Gateway® TurboGFP-N
  - A destination vector of choice
  - Invitrogen LR ClonaseTM II enzyme mix (Invitrogen Cat.# 11791-020)
  - Proteinase K solution (supplied with the LR ClonaseTM II enzyme mix)
  - TE-Buffer, pH 8.0 (10 mM Tris-HCl, pH 8.0, 1 mM EDTA)
  - Appropriate chemically competent E. coli host and growth media for expression
  - Appropriate selective plates.


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

attL1 site: 14-113
MCS: 117-194
Kozak translation initiation site: 195-205
TurboGFP: 202-900
attL2 site: 919-1018
Kanamycin resistance gene: 2236-3030
pUC origin of replication: 3615-4258


References:

  • 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:

TurboGFP-related materials (also referred to as "Products") are intended for research use only. The Products are covered by U.S. Pat. 7,678,893; European Pat. 1576157; 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.

Invitrogen Gateway® Technology: please see Limited Use Label License No. 19: Gateway® Cloning Products.

Copyright 2002-2018 Evrogen. All rights reserved.
Evrogen JSC, 16/10 Miklukho-Maklaya str., Moscow, Russia, Tel +7(495)988-4084, Fax +7(495)988-4085, e-mail:evrogen@evrogen.com