Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Antonio Ascue Avalos
Date created: 2011-09-20 11:00:00
Date modified: 2015-05-08 01:12:49
Low to medium copy Lambda Red recombineering compatible plasmid
| Types | DnaRegion |
| Roles | plasmid_vector Plasmid_Backbone |
| Sequences | BBa_K592202_sequence (Version 1) |
Description
BBa_K592202 is a BioBrick friendly vector with low to medium copy p15A replication origin (BBa_I50032) and a kanamycin antibiotic resistance marker, usable for Lambda Red recombineering in E coli. The E coli His operon terminator BBa_B0053 has been replaced with the late terminator of the Salmonella phage P22, and the kanamycin resistance marker is flanked by FRT sites so that the resistance marker can be removed after chromosomal integration using the FLP recombinase.Notes
To make the plasmid usable for Lambda Red recombineering, all sequences with homologies to the E coli chromosome had to be removed. To accomplish this, the E coli His operon terminator BBa_B0053 has been replaced with the late terminator of the Salmonella phage P22. To be able to remove the antibiotic resistance marker after chromosomal integration, the kanamycin marker from the pKD4 plasmid (Datsenko and Wanner, 2000) was used. The resistance marker can be removed after chromosomal integration using the FLP recombinase, leaving only the plasmid insert and a scar behind.Source
The P22 late terminator come from the genome of the Salmonella phage P22 (GenBank: AF217253.1, 19662..19702), and the kanamycin resistance marker flanked by FRT sites comes from the pKD4 plasmid (Datsenko and Wanner, 2000).| Sequence Annotation | Location | Component / Role(s) |
| L-red_R_primer Translational stop BBa_B0042 Terminator BBa_B0054 VR site BBa_G00102 Terminator BBa_B0062 p15A ori BBa_I50032 L-red_F_primer FRT site Kanamycin resistance marker FRT site P22 late terminator VF2 site BBa_G00100 T3 terminator BBa_B0055 Translational stop BBa_B0042 | 20,41 35,46 47,115 116,135 136,176 184,1748 1726,1745 1781,1826 2017,2811 3172,3219 3245,3285 3286,3305 3306,3383 3384,3395 | feature/primer_binding primer_binding_site stop_codon feature/stop sequence_feature feature/misc feature/primer_binding primer_binding_site feature/misc sequence_feature feature/misc sequence_feature feature/primer_binding primer_binding_site sequence_feature feature/misc feature/cds CDS sequence_feature feature/misc sequence_feature feature/misc feature/primer_binding primer_binding_site feature/misc sequence_feature feature/stop stop_codon |