Gateway® TagBFP-AS-C

Gateway® TagBFP-AS-C entry clone

cat.# FP177

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
Gateway® TagBFP-AS-CFP17720 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 typeGateway® entry clone
ReporterTagBFP
Reporter codon usageArabidopsis and Saccharomyces
Promoter for TagBFPNO
Host cellsprokaryotic
Selectionprokaryotic
ReplicationpUC ori
Use Generation of fusions to the C-terminus of TagBFP; transfer of the construct encoding TagBFP or its fusion into Gateway® destination vectors
Multiple cloning site (MCS)
TagBFPBgl IISac IEcoR ISal ISac IISma I/Xma IStops
BspE IXho IHind IIIPst IKpn IApa IBamH IAttL 2 site
...AAC.TCC.GGA.CTC.AGA.TCT.CGA.GCT.CAA.GCT.TCG.AAT.TCT.GCA.GTC.GAC.GGT.ACC.GCG.GGC.CCG.GGA.TCC.ACC.GGA.TCT.AGG.TAA.CTG.AAC.C...

Vector description

Gateway® TagBFP-AS-C entry clone is a vector containing blue fluorescent protein TagBFP gene variant with codon usage optimized for high expression in Arabidopsis and Saccharomyces (see reporter description). TagBFP 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 TagBFP gene into a number of Gateway® destination vectors for expression in different experimental systems. Multiple cloning site (MCS) located at the 3'-end of TagBFP gene allows to generate fusions to the TagBFP C-terminus for expression, localization and cellular dynamics studies.

To increase mRNA translation efficiency, Kozak consensus translation initiation site is generated upstream of the TagBFP 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 TagBFP 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 TagBFP C-terminus when inserted in the same reading frame as TagBFP and no in-frame stop codons are present.

Alternatively, TagBFP gene can be fused to the 3'-end of a gene of interest by LR recombination of the Gateway® TagBFP-AS-C with a destination vector containing this gene in a correct reading frame. In this case, the protein of interest will be expressed as a fusion to the TagBFP N-terminus.

TagBFP-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 TagBFP gene or TagBFP-fusion construct into the destination vector you will need:

  - Purified plasmid DNA of Gateway® TagBFP-AS-C
  - 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
Kozak translation initiation site: 129-139
TagBFP-AS: 136-834
MCS: 835-900
attL2 site: 920-1019
Kanamycin resistance gene: 2244-3038
pUC origin of replication: 3623-4266


References:

  • 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.

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

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