Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Patrick Rosendahl Andreassen
Date created: 2015-03-16 12:00:00
Date modified: 2015-05-08 01:09:06
GFP reporter with flexible linker at N-terminus for creation of GFP fusions
| Types | DnaRegion |
| Roles | Reporter engineered_region |
| Sequences | BBa_K1088052_sequence (Version 1) |
Description
This part will be suffixed to proteins to create protein-GFP fusions with a flexible linker in between to increase chances of both proteins to be functional.GFP fusions have variuos functions e.g. protein localization assays, immunoblotting and protein purification using anti-GFP antibodies. For protein purification, or other applications of seperating the fused proteins, a TEV protease recognition site is included in the flexible linker.
Note that standard RFC[10] assembly with a part suffixed to this part will result in a scar-site that includes an unwanted stop-codon. To prevent this, PCR amplification of inserts using appropriate primers is to be employed (see design page for details)
Notes
The linker was added according to step 1-4 as specified on the "Design" page of the flexible linkerUsing standard RFC[10] assembly of two parts creates scarsites (tactag). Transcription of the scarsite encodes: "uac uag" of which uag is a stop codon that would render the linker useless. Thus, standard assembly can not be employed for fusing proteins/domains.
1) Design primers for amplification of the N-terminal protein/domain that includes BamHI-site
a) Forward primer: 5'-cgctTCTAGAgNNN...NNN-3' - includes XbaI-site and sequence complementary to DNA sequence of N-terminal protein/domain
b) Reverse primer: 5'-atatGGATCCNNN...NNN-3' - includes BamHI-site and sequence complementary to DNA sequence of N-terminal protein/domain (OBS! do NOT include stop-codons of coding sequences)
2) PCR amplify BioBrick with designed primer
3) digest pSB1C3-Linker:GFP and PCR product with XbaI and BamHI
4) ligate the digested pSB1C3-Linker:GFP and PCR product to create a brick with your protein/domain fused with GFP
Source
BBa_I13401 and BBa_K1088051| Access | Instance | Definition |
| public public | BBa_K1088051 BBa_I13401 | Linker BBa_I13401 |
| Sequence Annotation | Location | Component / Role(s) |
| BBa_K1088051 BBa_I13401 | 1,30 31,887 | Linker BBa_I13401 |