GreenGate 2.0 Toolkit
(Kit #
1000000241
)
Depositing Lab: Alexis Maizel
The GreenGate 2.0 (GG2.0) toolkit enables the assembly and stacking of complex transcriptional units with the GreenGate Cloning System. This is backwards compatible with most existing GreenGate modules.
The main features of GG2.0 include:
- streamlining the generation of Level 0 modules by use of a Universal Entry Generator plasmid (pUEG).
- allowing the combination of up to 12 Level 0 modules in a Level 1 transcriptional unit.
- allowing the creation of several transcriptional units on the same plasmid (GreenBraid).
Additionally, GG2.0 includes a variety of pre-assembled vectors. Namely:
- Level 0 modules for multiplex expression of guide RNAs for CRISPR/Cas9 genome editing.
- Level 1 vectors for dexamethasone-inducible gene expression.
- Level 1 vectors for ubiquitous expression of plasma membrane and nuclear fluorescent markers.
This kit will be sent as bacterial glycerol stocks in 96-well plate format.
Original Publication
GreenGate 2.0: Backwards compatible addons for assembly of complex transcriptional units and their stacking with GreenGate. Piepers M, Erbstein K, Reyes-Hernandez J, Song C, Tessi T, Petrasiunaite V, Faerber N, Distel K, Maizel A. PLoS One. 2023 Sep 8;18(9):e0290097. doi: 10.1371/journal.pone.0290097. PubMed (Link opens in a new window) Article (Link opens in a new window).
Description
The 68 plasmids GG2.0 toolkit contains:
- a Universal Entry Generator plasmid (pUEG) for creating any Level 0 clones
- 21 empty Level 0 clones that expand the original GreenGate set by subdividing the GG A to E modules into 14 submodules (Bridging modules are provided for increased modularity.)
- 9 Level 0 plasmids for expressing one or two gRNAs from the pU3 or pU6 promoter
- 1 plasmid template for generating 2 gRNA insert
- 1 Level 0 (GreenGate B module) for zCas9i expression (insert: SpCas9_introns_2xNLS-Pea3A(T) term)
- 6 Level 1 braiding vectors
- 1 Level 1 vector allowing the pUBI10-driven expression of LhG4 (this can be used for braiding)
- 22 Level 1 destination vectors containing plasma membrane or nuclear fluorescent markers (all pUB10-driven) (these can be used for braiding)
- 6 Level 2 destination vectors for braiding of multiple constructs
Important: The kit contains no standard plant parts (promoters, tags, coding sequence, terminators, etc.). These are available as part of the GreenGate Cloning System.
- Figure 1. GreenGate 2.0 (GG2.0) expands the current GreenGate (GG) features set. GG2.0 offers more overhangs subdividing the existing GG A to E modules into 14 sub-modules. These modules can be cloned in a unique Universal entry vector to generate Level 0 entry clones. Up to 12 Level 0 clones can be assembled in one GG2.0 transcriptional unit (TU) compared to six for GG. Whereas GG only allowed the combination of two TUs to form a multi-gene Level 2 assembly, GG2.0 leverages GreenBraiding for the parallel, iterative assembly of a limitless number of TUs in Level 2 assemblies.
Plasmid Sequences
GreenGate2 Vectors (ZIP, 1.3 MB)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 GreenGate 2.0 Toolkit was a gift from Alexis Maizel (Addgene kit #1000000241).”
For your Reference section:
GreenGate 2.0: Backwards compatible addons for assembly of complex transcriptional units and their stacking with GreenGate. Piepers M, Erbstein K, Reyes-Hernandez J, Song C, Tessi T, Petrasiunaite V, Faerber N, Distel K, Maizel A. PLoS One. 2023 Sep 8;18(9):e0290097. doi: 10.1371/journal.pone.0290097. PubMed (Link opens in a new window) Article (Link opens in a new window).
GreenGate 2.0 Toolkit - #1000000241
- 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 | |
| Spectinomycin |
Inventory
| Well | Plasmid | Resistance |
|---|---|---|
| A / 1 | pR540 | Ampicillin |
| A / 2 | pR562 | Ampicillin |
| A / 3 | pAM745 | Ampicillin |
| A / 4 | pAM749 | Ampicillin |
| A / 5 | pAM750 | Ampicillin |
| A / 6 | pAM751 | Ampicillin |
| A / 7 | pAM752 | Ampicillin |
| A / 8 | pAM753 | Ampicillin |
| A / 9 | pAM754 | Ampicillin |
| A / 10 | pAM755 | Ampicillin |
| A / 11 | pAM756 | Ampicillin |
| A / 12 | pAM757 | Ampicillin |
| B / 1 | pAM758 | Ampicillin |
| B / 2 | pAM759 | Ampicillin |
| B / 3 | pAM760 | Ampicillin |
| B / 4 | pAM761 | Ampicillin |
| B / 5 | pAM762 | Ampicillin |
| B / 6 | pAM763 | Ampicillin |
| B / 7 | pAM764 | Ampicillin |
| B / 8 | pAM765 | Ampicillin |
| B / 9 | pAM766 | Ampicillin |
| B / 10 | pAM767 | Ampicillin |
| B / 11 | pAM768 | Ampicillin |
| B / 12 | pKE014 | Ampicillin |
| C / 1 | pCS017 | Ampicillin |
| C / 2 | pKE010 | Ampicillin |
| C / 3 | pCS018 | Ampicillin |
| C / 4 | pKE011 | Ampicillin |
| C / 5 | pCS019 | Ampicillin |
| C / 6 | pKE012 | Ampicillin |
| C / 7 | pCS020 | Ampicillin |
| C / 8 | pKE013 | Ampicillin |
| C / 9 | pCS021 | Ampicillin |
| C / 10 | pMP116 | Kanamycin |
| C / 11 | pMP120 | Kanamycin |
| C / 12 | pMP117 | Kanamycin |
| D / 1 | pMP121 | Kanamycin |
| D / 2 | pMP072 | Kanamycin |
| D / 3 | pMP073 | Kanamycin |
| D / 4 | pTT135 | Kanamycin |
| D / 5 | pMP107 | Kanamycin |
| D / 6 | pMP104 | Kanamycin |
| D / 7 | pMP105 | Kanamycin |
| D / 8 | pMP108 | Kanamycin |
| D / 9 | pMP065 | Kanamycin |
| D / 10 | pMP086 | Kanamycin |
| D / 11 | pKD013 | Kanamycin |
| D / 12 | pKD016 | Kanamycin |
| E / 1 | pKD014 | Kanamycin |
| E / 2 | pKD015 | Kanamycin |
| E / 3 | pKD011 | Kanamycin |
| E / 4 | pKD005 | Kanamycin |
| E / 5 | pKD012 | Kanamycin |
| E / 6 | pKD006 | Kanamycin |
| E / 7 | pKD009 | Kanamycin |
| E / 8 | pKD003 | Kanamycin |
| E / 9 | pKD010 | Kanamycin |
| E / 10 | pKD004 | Kanamycin |
| E / 11 | pKD007 | Kanamycin |
| E / 12 | pKD001 | Kanamycin |
| F / 1 | pKD008 | Kanamycin |
| F / 2 | pKD002 | Kanamycin |
| F / 3 | pMP118 | Spectinomycin |
| F / 4 | pMP122 | Spectinomycin |
| F / 5 | pMP119 | Spectinomycin |
| F / 6 | pMP123 | Spectinomycin |
| F / 7 | pMP074 | Spectinomycin |
| F / 8 | pMP075 | Spectinomycin |