Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Jean Cury
Date created: 2012-10-24 11:00:00
Date modified: 2015-05-08 01:13:46
P1003** Kan resistant gene with 2 Amber Codon
| Types | DnaRegion |
| Roles | engineered_region Translational_Unit |
| Sequences | BBa_K914018_sequence (Version 1) |
Description
The amber mutations avoid the expression of the P1003 gene even at usual concentration that prevent the expression of kanamycin resistance, although one mutation (BBa_K914009) permits some leakage in translation for this gene. This mutation is rescued with the tRNA amber suppressor supD (BBa_K914000). The idea here is to prevent the kanamycin gene resistance to be expressed in case horizontal gene transfer.Notes
I have need to design the primers that will introduce a mutation from a serine that was closer to the TAG codon (either 1, 2 or 3 mutations, here it was 1 and 2 mutations for both mutations), and it is necessary to have the amber codon in the middle of the coding sequence, not in the beginning, to be not interpreted as a alternative start codon for instance.Source
This part is made from the BBa_K914009 part from which I add a second mutation with the QuickChange Site-directed mutagenesis kit, Agilent.| Sequence Annotation | Location | Component / Role(s) |
| BBa_P1003 kanamycin resistance Ser133->Amber codon BBa_K914009 Ser203->Amber mutation | 1,967 149,967 546,546 1,967 609,610 | engineered_region feature/BioBrick feature/cds CDS point_mutation feature/s_mutation engineered_region feature/BioBrick feature/mutation sequence_alteration |