Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: George Chao, Misha Patel, Hannah Aho, Molly Swanson, Dana Morrone
Date created: 2012-10-02 11:00:00
Date modified: 2015-05-08 01:13:28
G418 selection yeast shuttle vector designed for BioBrick
| Types | DnaRegion |
| Roles | Plasmid_Backbone plasmid_vector |
| Sequences | BBa_K814015_sequence (Version 1) |
Description
A yeast/E. coli shuttle vector was designed with BioBrick prefix and suffix sites such that a yeast expression cassette could be inserted through basic BioBrick assembly. G418 was chosen instead of an auxotrophic marker for plasmid selection in yeast.To synthesize the overall plasmid backbone, PCR primers were designed to amplify five desired fragments (with 25-30 bp overlap) from different plasmids, which could be joined by Gibson Assembly:
1. BioBrick destination plasmid pSB1C3 containing the MCS and rep (pMB1).
2. BioBrick destination plasmid pSB1C3 containing the Chloramphenical resistance gene.
3. 2u Gibson Extract provided by the Schmidt-Dannert lab (University of Minnesota) containing the 2u ORI for yeast.
4. pkT127 provided by the Schmidt-Dannert lab containing G418 resistance genes and tTEF1.
5. ATCC plasmid provided by the Schmidt-Dannert lab containing the ADH1 promoter to drive the G418 R gene.
Notes
A number of mutations were directed so as to maintin the uniqueness of BioBrick cut sites.Source
This plasmid was assembled though Gibson assembly. Gibson assembly pieces were amplified from preexisting plasmid templates.| Sequence Annotation | Location | Component / Role(s) |
| BioBrick suffix His operon terminator rep (pBM1) T0 terminator Chloramphenicol resistance 2u Yeast ORI tTEF1 terminator G418 resistance marker Yeast Kozak pADH1 VR VF2 stem_loop | 1,20 21,91 260,614 926,1031 1043,1703 1803,3009 3035,3259 3260,4069 4070,4075 4095,5539 155,170 5565,5583 5654,5673 | sequence_feature feature/misc feature/stem_loop stem_loop feature/misc sequence_feature stem_loop feature/stem_loop feature/protein CDS sequence_feature feature/misc stem_loop feature/stem_loop feature/protein CDS feature/rbs ribosome_entry_site promoter feature/promoter feature/primer_binding primer_binding_site primer_binding_site feature/primer_binding stem_loop feature/stem_loop |