Gateway® Dendra2-At-N entry clone

cat.# FP825

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: Data sheet (pdf) Sequence (txt) Restriction map (pdf) Restriction table (pdf)

Product Cat.# Size Price Gateway® Dendra2-At-N FP825 20 μg € 400.00 The price does not include delivery. The price varies in different countries. Please contact your local distributor for exact prices and delivery information. Vector type Gateway® entry clone Reporter Dendra2 Reporter codon usage Arabidopsis Promoter for Dendra2 NO Host cells prokaryotic Selection kanamycin Replication pUC ori Use Generation of fusions to the N-terminus of Arabidopsis Dendra2 coding sequence; transfer of Dendra2 or its fusion into a Gateway® destination vector

Multiple cloning site (MCS)

attL1 site				Bgl II			Sac I		Hind III		EcoR I			Sal I		Kpn I			Apa I				BamH I		Age I		Dendra2
	Afe I				Xho I							Pst I					Sac II				Sma I/Xma 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.ACC.	ATG.AAC.ACT

Vector description

Gateway® Dendra2-At-N is a vector encoding green-to-red fluorescent protein Dendra2 (see protein description) flanked by attL1 and attL2 sites allowing site-specific recombination. The Invitrogen Gateway® Technology provides a rapid and highly efficient way to transfer the Dendra2 gene into a number of Gateway® expression vectors (destination vectors) for expression in different experimental systems. Multiple cloning site (MCS) located at the 5'-end of Dendra2 gene allows to generate fusions to the Dendra2 N-terminus for expression, localization and cellular dynamics studies.

Dendra2 codon usage is optimized for high expression in Arabidopsis. To increase mRNA translation efficiency, Kozak consensus translation initiation site is generated upstream of Dendra2 coding sequence [Kozak, 1987].

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

Generation of Dendra2-tagged fusions

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 Dendra2 N-terminus when inserted in the same reading frame as Dendra2 and no in-frame stop codons are present.

Alternatively, Dendra2 gene can be fused to the 3'-end of a gene of interest by LR recombination of the Gateway® Dendra2-At-N with a destination vector containing this gene in a correct reading frame.

Dendra2-tagged fusions retain fluorescent properties of the native protein allowing fusion localization in vivo.

LR site-specific recombination

Please refer to Invitrogen Gateway® Technology description for detailed instructions regarding to perform LR site-specific recombination reaction. In general, to transfer Dendra2 gene or Dendra2-fusion construct into the destination vector you will need:

  - Purified plasmid DNA of Gateway® Dendra2-At-N
  - A destination vector of choice
  - Invitrogen LR Clonase^TM II enzyme mix (Invitrogen Cat# 11791-020)
  - Proteinase K solution (supplied with the LR Clonase^TM 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
Dendra2: 202-891
attL2 site: 937-1036
Kanamycin resistance gene: 2254-3048
pUC origin of replication: 3633-4276