EasyClone-MarkerFree Vector Set
Depositing Lab: Irina Borodina
The EasyClone-MarkerFree Vector Set is a complete kit for the introduction of genes into 11 predetermined EasyClone Chomosomal loci. The kit comes complete with one Cas9 expression vector, 11 integrative vectors (one for each locus), 14 gRNA helper vectors (11 for single site targeting and 3 for triple site targeting), one empty vector for the insertion of gRNA expression cassettes, and 2 example vectors containing bidirectional promoters.
This kit will be sent as bacterial glycerol stocks in 96-well plate format.
EasyClone-MarkerFree: A vector toolkit for marker-less integration of genes into Saccharomyces cerevisiae via CRISPR-Cas9. Jessop-Fabre MM, Jakočiūnas T, Stovicek V, Dai Z, Jensen MK, Keasling JD, Borodina I. Biotechnol J. 2016 Aug 11.; doi: 10.1002/biot.201600147. PubMed PMID: 27166612.
The EasyClone-MarkerFree Vector Set is a complete kit for the introduction of genes into 11 predetermined EasyClone Chomosomal loci. These have been validated for stability and high expression. Each of these sites is located between essential genes and is thus not prone to being looped out of the chromosome. Each site can express up to two genes when combined with a bidirectional promoter (one on the sense and one on the antisense strand).
The kit comes complete with a Cas9 expression vector, 11 single site gRNAs, and 3 triple site gRNAs that guide the Cas9 to cut the chromosome in the specified chromosomal locations. These triple site gRNAs direct the Cas9 to cut in three separate loci, leading to the potential insertion of 6 genes per transformation (2 per locus). The double stranded break(s) are then repaired by the host cell using the native homologous recombination machinery, which incorporates the EasyClone-MarkerFree vector(s) into the target loci via double cross-over. This system is very efficient (90% correct clones with single insert, 60-70% with triple inserts) so there is no need for the insertion of markers into the host chromosome. This greatly speeds up the time to develop strains.
EasyClone-MarkerFree_manual.pdf 826.4 KB
How to Cite this Kit
These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which they were created, and include Addgene in the Materials and Methods of your future publications.
For your Materials and Methods section:
“EasyClone-MarkerFree Vector Set was a gift from Irina Borodina (Addgene kit #1000000098).”
For your Reference section:
EasyClone-MarkerFree: A vector toolkit for marker-less integration of genes into Saccharomyces cerevisiae via CRISPR-Cas9. Jessop-Fabre MM, Jakočiūnas T, Stovicek V, Dai Z, Jensen MK, Keasling JD, Borodina I. PubMed PMID: 27166612
EasyClone-MarkerFree Vector Set - #1000000098
- Resistance Color Key
Each circle corresponds to a specific antibiotic resistance in the kit plate map wells.
Searchable and sortable table of all plasmids in kit. The Well column lists the plasmid well location in its plate. The Plasmid column links to a plasmid's individual web page.
- Kit Plate Map
96-well plate map for plasmid layout. Hovering over a well reveals the plasmid name, while clicking on a well opens the plasmid page.
Resistance Color Key