pTurboRFP-PRL

pTurboRFP-PRL vector

cat.# FP235

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
pTurboRFP-PRLFP23520 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 typepromoterless expression vector
ReporterTurboRFP
Reporter codon usagemammalian
Promoter for TurboRFPNO
Host cellsmammalian, prokaryotic
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)
Bgl II Sac I Hind III EcoR I Sal I Kpn I Apa I BamH I Age I TurboRFP
Afe I Xho I Pst I Sac II Sma I/Xma I
ACT 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.ACC. ATG.AGC.GAG

Vector description

pTurboRFP-PRL is a promoterless vector encoding red (orange) fluorescent protein TurboRFP, which can be used as in vivo reporter of promoter activity (see reporter description). TurboRFP 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 TurboRFP coding sequence [Kozak, 1987].

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 TurboRFP.

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: 12-96
TurboRFP
Kozak consensus translation initiation site: 90-100
Start codon (ATG): 97-99
Stop codon: 790-792
SV40 early mRNA polyadenylation signal
Polyadenylation signals: 946-951 & 975-980
mRNA 3' ends: 984 & 996
f1 single-strand DNA origin: 1043-1498
Bacterial promoter for expression of Kanr gene
-35 region: 1560-1565
-10 region: 1583-1588
Transcription start point: 1595
SV40 origin of replication: 1839-1974
SV40 early promoter
Enhancer (72-bp tandem repeats): 1672-1743 & 1744-1815
21-bp repeats: 1819-1839, 1840-1860 & 1862-1882
Early promoter element: 1895-1901
Major transcription start points: 1891, 1929, 1935 & 1940
Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 2023-2025
Stop codon: 2815-2817
G->A mutation to remove Pst I site: 2205
C->A (Arg to Ser) mutation to remove BssH II site: 2551
Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 3053-3058 & 3066-3071
pUC plasmid replication origin: 3402-4045


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:

TurboRFP-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.

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