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    peTurboYFP-PRL-dest1

peTurboYFP-PRL-dest1 vector

cat.# FP616

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
peTurboYFP-PRL-dest1FP61620 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 typepromoterless expression vector
ReporterTurboYFP
Reporter codon usagemammalian
Promoter for TurboYFPNO
Host cellsmammalian
Selectionprokaryotic – kanamycin
eukaryotic – neomycin (G418)
Replicationprokaryotic – pUC ori
eukaryotic – SV40 ori
Use Monitoring of activity of different promoters and promoter/enhancer combinations
Multiple cloning site (MCS)
Afe IXho IHind IIIPst I*Kpn IApa IBamH I*TurboYFP-dest1
Bgl II*Sac IEcoR I*Sal ISac II*Sma I/Xma IAge I
...A.GCG.CTA.CCG.GAC.TCA.GAT.CTC.GAG.CTC.AAG.CTT.CGA.ATT.CTG.CAG.TCG.ACG.GTA.CCG.CGG.GCC.CGG.GAT.CCA.CCG.GTC.GCC.ACC.ATG.A...         

* – not unique site.

Vector description

peTurboYFP-PRL-dest1 is a promoterless vector encoding destabilized variant of the yellow fluorescent protein TurboYFP, which can be used as in vivo reporter of promoter activity (see reporter description). To generate TurboYFP-dest1 variant, residues 422-461 of mouse ornithine decarboxylase (MODC) were fused to the TurboYFP C-terminus. This MODC region contains a PEST amino acid sequence that targets the protein for degradation and provides for rapid protein turnover [Li et al., 1998]. TurboYFP-dest1 retains fluorescent properties of the native protein and has a half-life of approximately 1-1.5 hours, as measured by fluorescence intensity of cells treated with the protein synthesis inhibitor, cycloheximide. Rapid TurboYFP-dest1 turnover allows accurate analysis of changes in gene regulation.

TurboYFP-dest1 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 TurboYFP-dest1 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 TurboYFP-dest1 coding sequence in order to increase the protein expression level.

Multiple cloning site (MCS) is located upstream of the Kozak consensus translation initiation site and can be used to clone a promoter or a promoter/enchancer combination of interest. Without the addition of a functional promoter, this vector will not express TurboYFP-dest1.

The vector backbone contains 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.

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.


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

MCS: 12-89
TurboYFP-dest1
Kozak consensus translation initiation site: 90-100
Start codon (ATG): 97-99
Last amino acid in TurboYFP: 823-825
Amino acid residues of mouse ornithine decarboxylase (MODC) PEST sequence: 847-966
Stop codon: 967-969
Fragment of human beta globin (HBB) gene
Last 35 bp of HBB exon 2: 978-1012
HBB intron 2: 974-1859
First 233 bp of HBB exon 3: 1860-2096
SV40 early mRNA polyadenylation signal
Polyadenylation signals: 2238-2243 & 2267-2272
mRNA 3' ends: 2276 & 2288
f1 single-strand DNA origin: 2335-2790
Bacterial promoter for expression of Kanr gene
-35 region: 2852-2857
-10 region: 2875-2880
Transcription start point: 2887
SV40 origin of replication: 3131-3266
SV40 early promoter
Enhancer (72-bp tandem repeats): 2964-3035 & 3036-3107
21-bp repeats: 3111-3131, 3132-3152 & 3154-3174
Early promoter element: 3187-3193
Major transcription start points: 3183, 3221, 3227 & 3232
Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 3315-3317
Stop codon: 4107-4109
G->A mutation to remove Pst I site: 3497
C->A (Arg to Ser) mutation to remove BssH II site: 3843
Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 4345-4350 & 4358-4363
pUC plasmid replication origin: 4694-5337


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
  • Li X, Zhao X, Fang Y, Jiang X, Duong T, Fan C, Huang CC, Kain SR. Generation of destabilized green fluorescent protein as a transcription reporter. J Biol Chem. 1998; 273 (52):34970-5. / pmid: 9857028

Notice to Purchaser:

TurboYFP-related materials (also referred to as "Products") are intended for research use only. The Products are covered by U.S. Pat. 7,951,923; European Pat. 03779067; 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.

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