pTagBFP-N

pTagBFP-N vector

cat.# FP172

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:
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pTagBFP-NFP17220 μg€ 400 / 200*
*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
ReporterTagBFP
Reporter codon usagemammalian
Promoter for TagBFPPCMV IE
Host cellsmammalian
Selectionprokaryotic – kanamycin
eukaryotic – neomycin (G418)
Replicationprokaryotic – pUC ori
eukaryotic – SV40 ori
Use TagBFP expression in mammalian cells; generation of fusions to the TagBFP N-terminus
Multiple cloning site (MCS)
Nhe IBgl IISac IEcoR ISal ISac IISma I/Xma IAge ITagBFP
Afe IXho IHind III*Pst IKpn IApa IBamH I
GCTA.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...

* – not unique site.

Vector description

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

TagBFP 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 TagBFP coding sequence [Kozak, 1987]. Multiple cloning site (MCS) is located between PCMV IE and TagBFP 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 TagBFP 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 TagBFP N-terminus when inserted in the same reading frame as TagBFP and no in-frame stop codons are present. The inserted sequence should contain an initiating ATG codon. TagBFP-tagged fusions retain fluorescent properties of the native protein allowing fusion localization in vivo. Unmodified vector will express TagBFP 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

pTagBFP-N vector can be transfected into mammalian cells by any known transfection method. CMV promoter provides strong, constitutive expression of TagBFP 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
TagBFP
Kozak consensus translation initiation site: 672-682
Start codon (ATG): 679-681
Stop codon: 1378-1380
SV40 early mRNA polyadenylation signal
Polyadenylation signals: 1534-1539 & 1563-1568
mRNA 3' ends: 1572 & 1584
f1 single-strand DNA origin: 1631-2086
Bacterial promoter for expression of Kanr gene
-35 region: 2148-2153
-10 region: 2171-2176
Transcription start point: 2183
SV40 origin of replication: 2427-2562
SV40 early promoter
Enhancer (72-bp tandem repeats): 2260-2331 & 2332-2403
21-bp repeats: 2407-2427, 2428-2448 & 2450-2470
Early promoter element: 2483-2489
Major transcription start points: 2479, 2517, 2523 & 2528
Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 2611-2613
Stop codon: 3403-3405
G->A mutation to remove Pst I site: 2793
C->A (Arg to Ser) mutation to remove BssH II site: 3139
Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 3641-3646 & 3654-3659
pUC plasmid replication origin: 3990-4633


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:

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