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Markerless Yeast Localization and Overexpression (MyLO) CRISPR-Cas9 Toolkit
(Kit # 1000000210 )

Depositing Lab:   Vincent Martin

The aim of the MyLO CRISPR-Cas9 toolkit is to improve efficiency, and decrease complexity, of common markerless manipulations of the yeast genome. This is achieved with a suite of vectors with Cas9 and either complete guides or guide cloning sites with GFP dropout cassettes, which facilitate transformation-based cloning or Golden Gate cloning. The Golden Gate cloning Cas9 vectors contain novel split selection cassettes that improve transformation efficiencies. The kit also includes parent vectors for Golden Gate cloning target genes into integration cassettes with either N- or C-terminal GFP, RFP, or 3xHA tags. Finally, a suite of integration cassettes featuring validated GFP- or RFP-tagged compartmental markers is included.

This kit will be sent as bacterial glycerol stocks in 96-well plate format.

Add to Cart
$ 400 USD + shipping

Available to academics and nonprofits only.

Original Publication

The MyLO CRISPR-Cas9 toolkit: a markerless yeast localization and overexpression CRISPR-Cas9 toolkit. Bean BDM, Whiteway M, Martin VJJ. G3 (Bethesda). 2022 Jul 29;12(8):jkac154. doi: 10.1093/g3journal/jkac154. PubMed (Link opens in a new window) Article (Link opens in a new window)

Description

This toolkit contains Cas9-gRNA vectors, integration plasmids, and organellar localization markers as a versatile set of plasmids for Cas9-mediated integration of tagged genes and markers into the yeast genome. The Cas9-gRNA vectors contain one of five selectable markers (KanR, HygR, NatR, LEU2, URA3). gRNAs are introduced through transformation-based cloning into vectors with full selection markers, Golden Gate cloning into vectors with novel split selection markers that improve transformation efficiencies, or using pre-cloned gRNA targeting seven common sites. A set of Golden Gate cloning-compatible plasmids allow quick production of integration cassettes for a gene of interest (GOI) with three different N- or C-terminal tags (GFP, RFP, HA) at three expression levels. These cassettes have multi-target homology arms targeting seven different safe harbor sites. This toolkit also includes a validated set of 21 integration-ready GFP- or RFP-tagged organellar localization markers for use in colocalization studies.

  • Overview of the three primary elements of the MyLO CRISPR-Cas9 toolkit

Kit-Specific Resources

Toolkit details, instructions, and recommended protocols are available in Supplemental File S2 of the original publication (PDF, 3.4 MB) (Link opens in a new window)

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:

“The Markerless Yeast Localization and Overexpression (MyLO) CRISPR-Cas9 Toolkit was a gift from Vincent Martin (Addgene kit #1000000210).”

For your Reference section:

The MyLO CRISPR-Cas9 toolkit: a markerless yeast localization and overexpression CRISPR-Cas9 toolkit. Bean BDM, Whiteway M, Martin VJJ. G3 (Bethesda). 2022 Jul 29;12(8):jkac154. doi: 10.1093/g3journal/jkac154. PubMed (Link opens in a new window) Article (Link opens in a new window)

The Markerless Yeast Localization and Overexpression (MyLO) CRISPR-Cas9 Toolkit - #1000000210

Resistance Color Key

Each circle corresponds to a specific antibiotic resistance in the kit plate map wells.

Inventory

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

Ampicillin
Kanamycin
Hygromycin

Inventory

Well Plasmid Resistance
A / 1 pBBK01 pCAS-Tyr-[gRNA: BsaI GFP dropout] (KanR, AmpR)
Ampicillin
A / 2 pBBK02 pCAS-Tyr-[gRNA: BsaI GFP dropout] (SplitKanR, AmpR)
Ampicillin
A / 3 pBBK03 pCAS-Tyr-[gRNA: BsaI GFP dropout] (HygR,AmpR)
Ampicillin
A / 4 pBBK04 pCAS-Tyr-[gRNA: BsaI GFP dropout] (SplitHygR,AmpR)
Ampicillin
A / 5 pBBK05 pCAS-Tyr-[gRNA: BsaI GFP dropout] (NatR,AmpR)
Ampicillin
A / 6 pBBK06 pCAS-Tyr-[gRNA: BsaI GFP dropout] (SplitNatR,AmpR)
Ampicillin
A / 7 pBBK07 pCAS-Tyr-[gRNA: BsaI GFP dropout] (LEU2,AmpR)
Ampicillin
A / 8 pBBK08 pCAS-Tyr-[gRNA: BsaI GFP dropout] (SplitLEU2,AmpR)
Ampicillin
A / 9 pBBK09 pCAS-Tyr-[gRNA: BsaI GFP dropout] (BsmBI-flanked URA3, AmpR)
Ampicillin
A / 10 pBBK10 pCAS-Tyr-[gRNA: BsaI GFP dropout] (SplitURA3,AmpR)
Ampicillin
A / 11 pBBK11 pCAS-Tyr-[gRNA: 1=FF18] (SplitKanR, AmpR)
Ampicillin
A / 12 pBBK12 pCAS-Tyr-[gRNA: 2=FF21] (SplitKanR, AmpR)
Ampicillin
B / 1 pBBK13 pCAS-Tyr-[gRNA: 3=XII-2] (SplitKanR, AmpR)
Ampicillin
B / 2 pBBK14 pCAS-Tyr-[gRNA: 4=XII-5] (SplitKanR, AmpR)
Ampicillin
B / 3 pBBK15 pCAS-Tyr-[gRNA: 5=ARS308] (SplitKanR, AmpR)
Ampicillin
B / 4 pBBK16 pCAS-Tyr-[gRNA: 6=ARS416] (SplitKanR, AmpR)
Ampicillin
B / 5 pBBK17 pCAS-Tyr-[gRNA: 7=HIS3] (SplitKanR, AmpR)
Ampicillin
B / 6 pBBK18 pCAS-Tyr-[gRNA: 1=FF18] (SplitHygR, AmpR)
Ampicillin
B / 7 pBBK19 pCAS-Tyr-[gRNA: 2=FF21] (SplitHygR, AmpR)
Ampicillin
B / 8 pBBK20 pCAS-Tyr-[gRNA: 3=XII-2] (SplitHygR, AmpR)
Ampicillin
B / 9 pBBK21 pCAS-Tyr-[gRNA: 4=XII-5] (SplitHygR, AmpR)
Ampicillin
B / 10 pBBK22 pCAS-Tyr-[gRNA: 5=ARS308] (SplitHygR, AmpR)
Ampicillin
B / 11 pBBK23 pCAS-Tyr-[gRNA: 6=ARS416] (SplitHygR, AmpR)
Ampicillin
B / 12 pBBK24 pCAS-Tyr-[gRNA: 7=HIS3] (SplitHygR, AmpR)
Ampicillin
C / 1 pBBK25 pHomL-TDH3p-Neon(gfp)-Link-GFPdropout-SSA1t-HomR (AmpR)
Ampicillin
C / 2 pBBK26 pHomL-TEF2p-Neon(gfp)-Link-GFPdropout-SSA1t-HomR (AmpR)
Ampicillin
C / 3 pBBK27 pHomL-RPL18Bp-Neon(gfp)-Link-GFPdropout-SSA1t-HomR (AmpR)
Ampicillin
C / 4 pBBK28 pHomL-TDH3p-GFPdropout-Link-Neon(gfp)-SSA1t-HomR (AmpR)
Ampicillin
C / 5 pBBK29 pHomL-TEF2p-GFPdropout-Link-Neon(gfp)-SSA1t-HomR (AmpR)
Ampicillin
C / 6 pBBK30 pHomL-RPL18Bp-GFPdropout-Link-Neon(gfp)-SSA1t-HomR (AmpR)
Ampicillin
C / 7 pBBK31 pHomL-TDH3p-Scarlet(rfp)-Link-GFPdropout-SSA1t-HomR (AmpR)
Ampicillin
C / 8 pBBK32 pHomL-TEF2p-Scarlet(rfp)-Link-GFPdropout-SSA1t-HomR (AmpR)
Ampicillin
C / 9 pBBK33 pHomL-RPL18Bp-Scarlet(rfp)-Link-GFPdropout-SSA1t-HomR (AmpR)
Ampicillin
C / 10 pBBK34 pHomL-TDH3p-GFPdropout-Link-Scarlet(rfp)-SSA1t-HomR (AmpR)
Ampicillin
C / 11 pBBK35 pHomL-TEF2p-GFPdropout-Link-Scarlet(rfp)-SSA1t-HomR (AmpR)
Ampicillin
C / 12 pBBK36 pHomL-RPL18Bp-GFPdropout-Link-Scarlet(rfp)-SSA1t-HomR (AmpR)
Ampicillin
D / 1 pBBK37 pHomL-TDH3p-3HA-Link-GFPdropout-SSA1t-HomR (AmpR)
Ampicillin
D / 2 pBBK38 pHomL-TEF2p-3HA-Link-GFPdropout-SSA1t-HomR (AmpR)
Ampicillin
D / 3 pBBK39 pHomL-RPL18Bp-3HA-Link-GFPdropout-SSA1t-HomR (AmpR)
Ampicillin
D / 4 pBBK40 pHomL-TDH3p-GFPdropout-Link-3HA-SSA1t-HomR (AmpR)
Ampicillin
D / 5 pBBK41 pHomL-TEF2p-GFPdropout-Link-3HA-SSA1t-HomR (AmpR)
Ampicillin
D / 6 pBBK42 pHomL-RPL18Bp-GFPdropout-Link-3HA-SSA1t-HomR (AmpR)
Ampicillin
D / 7 pBBK43 pHomL-TDH3p-GFPdropout-SSA1t-HomR (AmpR)
Ampicillin
D / 8 pBBK44 pHomL-TEF2p-GFPdropout-SSA1t-HomR (AmpR)
Ampicillin
D / 9 pBBK45 pHomL-RPL18Bp-GFPdropout-SSA1t-HomR (AmpR)
Ampicillin
D / 10 pBBK46 pHomL-RPL18Bp-RPC19-Link-Neon(gfp)-SSA1t-HomR (AmpR)
Ampicillin
D / 11 pBBK47 pHomL-RPL18Bp-SRM1-Link-Neon(gfp)-SSA1t-HomR (AmpR)
Ampicillin
D / 12 pBBK48 pHomL-RPL18Bp-SPC42-Link-Neon(gfp)-SSA1t-HomR (AmpR)
Ampicillin
E / 1 pBBK49 pHomL-RPL18Bp-Neon(gfp)-Link-TUB1-SSA1t-HomR (AmpR)
Ampicillin
E / 2 pBBK50 pHomL-RPL18Bp-ARC35-Link-Neon(gfp)-SSA1t-HomR (AmpR)
Ampicillin
E / 3 pBBK51 pHomL-RPL18Bp-Neon(gfp)-Link-SCS2tmh-SSA1t-HomR (AmpR)
Ampicillin
E / 4 pBBK52 pHomL-RPL18Bp-Neon(gfp)-Link-SED5-SSA1t-HomR (AmpR)
Ampicillin
E / 5 pBBK53 pHomL-RPL18Bp-Neon(gfp)-Link-SFT2-SSA1t-HomR (AmpR)
Ampicillin
E / 6 pBBK54 pHomL-RPL18Bp-Neon(gfp)-Link-2xPH(PLCd)-SSA1t-HomR (AmpR)
Ampicillin
E / 7 pBBK55 pHomL-RPL18Bp-PIL1-Link-Neon(gfp)-SSA1t-HomR (AmpR)
Ampicillin
E / 8 pBBK56 pHomL-RPL18Bp-Neon(gfp)-Link-VPS21-SSA1t-HomR (AmpR)
Ampicillin
E / 9 pBBK57 pHomL-RPL18Bp-SNF7-Link-Neon(gfp)-SSA1t-HomR (AmpR)
Ampicillin
E / 10 pBBK58 pHomL-RPL18Bp-Neon(gfp)-Link-YPT7-SSA1t-HomR (AmpR)
Ampicillin
E / 11 pBBK59 pHomL-RPL18Bp-Neon(gfp)-Link-VAM3-SSA1t-HomR (AmpR)
Ampicillin
E / 12 pBBK60 pHomL-RPL18Bp-PRC1-Link-Neon(gfp)-SSA1t-HomR (AmpR)
Ampicillin
F / 1 pBBK61 pHomL-RPL18Bp-Neon(gfp)-Link-ATG8-SSA1t-HomR (AmpR)
Ampicillin
F / 2 pBBK62 pHomL-RPL18Bp-ERG6-Link-Neon(gfp)-SSA1t-HomR (AmpR)
Ampicillin
F / 3 pBBK63 pHomL-RPL18Bp-PDR16-Link-Neon(gfp)-SSA1t-HomR (AmpR)
Ampicillin
F / 4 pBBK64 pHomL-RPL18Bp-PEX25-Link-Neon(gfp)-SSA1t-HomR (AmpR)
Ampicillin
F / 5 pBBK65 pHomL-RPL18Bp-preCOX4-Link-Neon(gfp)-SSA1t-HomR (AmpR)
Ampicillin
F / 6 pBBK66 pHomL-RPL18Bp-TOM70-Link-Neon(gfp)-SSA1t-HomR (AmpR)
Ampicillin
F / 7 pBBK67 pHomL-RPL18Bp-RPC19-Link-Scarlet(rfp)-SSA1t-HomR (AmpR)
Ampicillin
F / 8 pBBK68 pHomL-RPL18Bp-SRM1-Link-Scarlet(rfp)-SSA1t-HomR (AmpR)
Ampicillin
F / 9 pBBK69 pHomL-RPL18Bp-SPC42-Link-Scarlet(rfp)-SSA1t-HomR (AmpR)
Ampicillin
F / 10 pBBK70 pHomL-RPL18Bp-Scarlet(rfp)-Link-TUB1-SSA1t-HomR (AmpR)
Ampicillin
F / 11 pBBK71 pHomL-RPL18Bp-ARC35-Link-Scarlet(rfp)-SSA1t-HomR (AmpR)
Ampicillin
F / 12 pBBK72 pHomL-RPL18Bp-Scarlet(rfp)-Link-SCS2tmh-SSA1t-HomR (AmpR)
Ampicillin
G / 1 pBBK73 pHomL-RPL18Bp-Scarlet(rfp)-Link-SED5-SSA1t-HomR (AmpR)
Ampicillin
G / 2 pBBK74 pHomL-RPL18Bp-Scarlet(rfp)-Link-SFT2-SSA1t-HomR (AmpR)
Ampicillin
G / 3 pBBK75 pHomL-RPL18Bp-Scarlet(rfp)-Link-2xPH(PLCd)-SSA1t-HomR (AmpR)
Ampicillin
G / 4 pBBK76 pHomL-RPL18Bp-PIL1-Link-Scarlet(rfp)-SSA1t-HomR (AmpR)
Ampicillin
G / 5 pBBK77 pHomL-RPL18Bp-Scarlet(rfp)-Link-VPS21-SSA1t-HomR (AmpR)
Ampicillin
G / 6 pBBK78 pHomL-RPL18Bp-SNF7-Link-Scarlet(rfp)-SSA1t-HomR (AmpR)
Ampicillin
G / 7 pBBK79 pHomL-RPL18Bp-Scarlet(rfp)-Link-YPT7-SSA1t-HomR (AmpR)
Ampicillin
G / 8 pBBK80 pHomL-RPL18Bp-Scarlet(rfp)-Link-VAM3-SSA1t-HomR (AmpR)
Ampicillin
G / 9 pBBK81 pHomL-RPL18Bp-PRC1-Link-Scarlet(rfp)-SSA1t-HomR (AmpR)
Ampicillin
G / 10 pBBK82 pHomL-RPL18Bp-Scarlet(rfp)-Link-ATG8-SSA1t-HomR (AmpR)
Ampicillin
G / 11 pBBK83 pHomL-RPL18Bp-ERG6-Link-Scarlet(rfp)-SSA1t-HomR (AmpR)
Ampicillin
G / 12 pBBK84 pHomL-RPL18Bp-PDR16-Link-Scarlet(rfp)-SSA1t-HomR (AmpR)
Ampicillin
H / 1 pBBK85 pHomL-RPL18Bp-PEX25-Link-Scarlet(rfp)-SSA1t-HomR (AmpR)
Ampicillin
H / 2 pBBK86 pHomL-RPL18Bp-preCOX4-Link-Scarlet(rfp)-SSA1t-HomR (AmpR)
Ampicillin
H / 3 pBBK87 pHomL-RPL18Bp-TOM70-Link-Scarlet(rfp)-SSA1t-HomR (AmpR)
Ampicillin
H / 4 pBBK88 pHomL-TDH3p-Neon(gfp)-Link-2xPH(PLCd)-SSA1t-HomR (AmpR)
Ampicillin
H / 5 pBBK89 pHomL-TDH3p-Scarlet(rfp)-Link-2xPH-SSA1t-HomR (AmpR)
Ampicillin
H / 6 pBBK90 pHomL-TEF2p-Scarlet(rfp)-Link-2xPH-SSA1t-HomR (AmpR)
Ampicillin
H / 7 pBBK91 pHomL-TDH3p-Neon(gfp)-Link-SCS2tmh-SSA1t-HomR (AmpR)
Ampicillin
H / 8 pBBK92 pHomL-TDH3p-Scarlet(rfp)-Link-SCS2tmh-SSA1t-HomR (AmpR)
Ampicillin
H / 9 pBBK93 pHomL-TDH3p-Neon(gfp)-SSA1t-HomR (AmpR)
Ampicillin
H / 10 pBBK94 pCAS-Tyr-[gRNA: 2xBsaI+NotI cut sites] (KanR)
Kanamycin
H / 11 pBBK95 pCAS-Tyr-[gRNA: 2xBsaI+NotI cut sites] (HygR)
Hygromycin
H / 12 pBBK96 pHomL-ConLS-GFPdropout-ConR1-HomR (AmpR)
Ampicillin
Data calculated @ 2024-02-26

Kit Plate Map - #1000000210

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