Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Jeff Nivala
Date created: 2009-10-17 11:00:00
Date modified: 2015-05-08 01:11:30
Secretion Signal and Streptavidin Binding Tags on GFP
| Types | DnaRegion |
| Roles | Coding CDS |
| Sequences | BBa_K215012_sequence (Version 1) |
Description
K215002 drives the expression of BBa_K215001 by placing it under the control of a strong, IPTG-inducible promoter (R0011) and RBS (R0034). Any favorite protein (afp) can be inserted into a composite tag BBa_K215001. The composite tag has an NheI site (compatible with XbaI and SpeI, but make sure the resulting product stays in frame, described below) which places the afp in between a series of tags which can be used with the University of Washington 2009 Ideal Protein Purification system (IPP). The series of tags are, respectively: a Nano-Tag (binds to streptavidin), a 6x His tag (for IMAC purification), a TEV cute site (for removal of N-terminal tags), the NheI site (insertion of afp), TEV, 6x His tag, and the 181 C-terminal amino acids of prtB (secretion tag recognized by the secretion system: BBa_K215107). In order for the secretion tag to signal the secretion of afp the BioBrick BBa_K215107 must be present as well.Cutting a BioBrick coding sequence at X and S and pasting into the NheI site of this construct would result in a stop codon at the resulting scar site. Therefore, to insert a gene into the K215001 of this construct, a PCR product must be generated that keeps the gene of interest in frame with the rest of the sequence. To do this: