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URL: http://www.addgene.org/40236

Proper Citation: RRID:Addgene_40236

Insert Name: LOV-ipaA

Organism: Synthetic

Bacterial Resistance: Ampicillin

Defining Citation: PMID:22520757

Vector Backbone Description: Backbone Marker:EMD Biosciences; Backbone Size:5442; Vector Backbone:pET-21b(+); Vector Types:Bacterial Expression; Bacterial Resistance:Ampicillin

Comments: This vector encodes a photo-activable caged form of the vinculin binding ipaA peptide. Regarding the hybridized region, the first ten residues of the ipaA VBS1 helical peptide were identified as a close match to the last ten residues of the AsLOV2 Jα helix; five of the ten positions are identical, and the hydrophobic residues on the Jα helix that make critical contacts with the AsLOV2 domain beta-sheet at residues 539, 542, and 543 are conserved in the alignment with ipaA. Additionally, residues in the ipaA sequence that make extensive contacts with vinculin are conserved in the alignment (Ile 612, Ala 615, Ala 616, and Val 619 in ipaA). Side-chain optimization simulations were used to thread the first ten residues of ipaA onto the last ten residues of the Jα helix. The 540 position on the Jα helix was converted to isoleucine. The LOV-ipaA gene was synthesized with a six histidine N-terminal tag (Genscript, Piscataway, NJ, USA) and cloned into the pET21b vector. Protein coding sequence of LOV-ipaA: MHHHHHHGSLATTLERIEKNFVITDPRLPDNPIIFASDSFLQLTEYSREEILGRNCRFLQGPETDRATVRKIRDAIDNQTEVTVQLINYTKSGKKFWNLFHLQPMRDQKGDVQYFIGVQLDGTEHVRDAAEREGVMLIKKTANNIIKAAKDVTTSLSKVLKNIN

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