GreenGate Cloning System
(Kit #
1000000036
)
Depositing Lab: Jan Lohmann
The 56 plasmid GreenGate cloning kit can be used to create plant expression vectors containing several cassettes and generate multi-construct transgenic plants.
This kit consists of one 96-well plate, and will be shipped as bacterial glycerol stocks on dry ice. Samples should be frozen at -80°C immediately upon arrival. Individual plasmids can be ordered from each plasmid page and will be shipped as bacterial stabs.
Original Publication
GreenGate - A Novel, Versatile, and Efficient Cloning System for Plant Transgenesis. Lampropoulos A, Sutikovic Z, Wenzl C, Maegele I, Lohmann JU, Forner J. PLoS One 2013 Dec 20;8(12):e83043. doi: 10.1371/journal.pone.0083043. PubMed PMID 24376629
Description
GreenGate is a simple and efficient cloning system for rapidly assembling plant transformation constructs . It is based on the Golden Gate method. Using the type IIS restriction endonuclease BsaI and T4 DNA ligase, ready-to-use plant transformation vectors are built from six types of pre-cloned insert modules and a destination vector in a one-pot reaction. The six module types are (1) plant promoter, (2) N-terminal tag, (3) coding sequence (i.e. the gene of interest), (4) C-terminal tag, (5) plant terminator and (6) plant resistance cassette.
Learn more about the GreenGate cloning system by reading Addgene's blog post - Quick, Versatile Plant Transgenesis with GreenGate Plasmids.
GreenGate vector design and layout
A) The GreenGate cloning system uses six different types of pUC19 based entry vectors into which the single elements are inserted and a pGreen-IIS based destination vector. Magenta scissors depict the BsaI recognition sites. In each GreenGate reaction, six modules are ligated between the left border (LB) and the right border (RB) sequences of the destination vector yielding a ready-to-use plant transformation vector with an expression cassette and a resistance cassette. These six modules encompass a plant promoter, an N-terminal tag, a coding sequence (i.e. the gene of interest), a C-terminal tag, a plant terminator and a plant resistance cassette for selection of transgenic plants. The modules can only be ligated in this pre-defined order. B) The orderly assembly is enabled by a set of seven different overhangs. Each module is flanked at its 5’-end by the same overhang as the 3’-end of its preceding neighbor. The individual overhangs all differ from each other (and each other’s and their own reverse complement) by at least two out of the four nucleotides. This ensures that they can anneal only to the complementary overhang from the neighboring module in the right orientation. The underlined nucleotides define coding triplets to which all other elements have to be in frame.
C) The entry vectors are pUC19 based. The multiple cloning site of pUC19 has been replaced by two BsaI recognition sites (magenta scissors), the respective overhangs for each module type and a counter-selectable ccdB gene. PCR products can also be cloned via the BamHI and KpnI sites or via A-overhangs after XcmI digestion. Plac = lac promoter, SP6 = SP6 promoter, caR = chloramphenicol acetyltransferase gene, T7 = T7 promoter, lacZ = lacZa coding sequence, ampR = beta-lactamase gene, ori = origin of replication. D) The destination vectors are pGreen-IIS based. A counter-selectable ccdB-cassette has been inserted between the LB and RB sequences, flanked by BsaI sites, with overhangs A and G. promoter = bacterial promoter. The pSa origin of replication (ori A. tum.) requires the presence of the helper plasmid pSOUP in agrobacteria.
Kit Documentation
When receiving kit as frozen glycerol stocks on dry ice:
GreenGate cloning Kit 264.4 KBResources
More information on these plasmids and their use can be found in the original publication.
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 plasmid kit used for generation of plant transformation constructs was a gift from Jan Lohmann (Addgene kit # 1000000036)"
For your Reference section:
GreenGate - A Novel, Versatile, and Efficient Cloning System for Plant Transgenesis. Lampropoulos A, Sutikovic Z, Wenzl C, Maegele I, Lohmann JU, Forner J. PLoS One 2013 Dec 20;8(12):e83043. doi: 10.1371/journal.pone.0083043. PubMed PMID 24376629
GreenGate - #1000000036
- 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 | |
| Chloramphenicol and Ampicillin | |
| Chloramphenicol and Gentamicin | |
| Chloramphenicol and Kanamycin | |
| Chloramphenicol and Spectinomycin |
Inventory
| Well | Plasmid | Resistance |
|---|---|---|
| A / 1 | pGGA002 | Ampicillin |
| A / 2 | pGGA003 | Ampicillin |
| A / 3 | pGGA004 | Ampicillin |
| A / 4 | pGGA006 | Ampicillin |
| A / 5 | pGGA008 | Ampicillin |
| A / 6 | pGGA012 | Ampicillin |
| A / 7 | pGGB001 | Ampicillin |
| A / 8 | pGGB002 | Ampicillin |
| A / 9 | pGGB003 | Ampicillin |
| A / 10 | pGGB005 | Ampicillin |
| A / 11 | pGGB006 | Ampicillin |
| A / 12 | pGGC001 | Ampicillin |
| B / 1 | pGGC011 | Ampicillin |
| B / 2 | pGGC012 | Ampicillin |
| B / 3 | pGGC014 | Ampicillin |
| B / 4 | pGGC015 | Ampicillin |
| B / 5 | pGGC024 | Ampicillin |
| B / 6 | pGGC025 | Ampicillin |
| B / 7 | pGGC026 | Ampicillin |
| B / 8 | pGGC051 | Ampicillin |
| B / 9 | pGGD001 | Ampicillin |
| B / 10 | pGGD002 | Ampicillin |
| B / 11 | pGGD003 | Ampicillin |
| B / 12 | pGGD006 | Ampicillin |
| C / 1 | pGGD007 | Ampicillin |
| C / 2 | pGGD008 | Ampicillin |
| C / 3 | pGGE001 | Ampicillin |
| C / 4 | pGGE002 | Ampicillin |
| C / 5 | pGGE009 | Ampicillin |
| C / 6 | pGGF001 | Ampicillin |
| C / 7 | pGGF002 | Ampicillin |
| C / 8 | pGGF003 | Ampicillin |
| C / 9 | pGGF004 | Ampicillin |
| C / 10 | pGGF005 | Ampicillin |
| C / 11 | pGGF007 | Ampicillin |
| C / 12 | pGGF008 | Ampicillin |
| D / 1 | pGGF012 | Ampicillin |
| D / 2 | pGGG001 | Ampicillin |
| D / 3 | pGGG002 | Ampicillin |
| D / 4 | pGGD005 | Ampicillin |
| D / 5 | pGGE003 | Ampicillin |
| D / 6 | pGGE005 | Ampicillin |
| D / 7 | pGGA000 | Chloramphenicol and Ampicillin |
| D / 8 | pGGB000 | Chloramphenicol and Ampicillin |
| D / 9 | pGGC000 | Chloramphenicol and Ampicillin |
| D / 10 | pGGD000 | Chloramphenicol and Ampicillin |
| D / 11 | pGGE000 | Chloramphenicol and Ampicillin |
| D / 12 | pGGF000 | Chloramphenicol and Ampicillin |
| E / 1 | pGGH000 | Chloramphenicol and Ampicillin |
| E / 2 | pGGI000 | Chloramphenicol and Ampicillin |
| E / 3 | pGGY003 | Chloramphenicol and Gentamicin |
| E / 4 | pGGY001 | Chloramphenicol and Gentamicin |
| E / 5 | pGGM000 | Chloramphenicol and Kanamycin |
| E / 6 | pGGN000 | Chloramphenicol and Kanamycin |
| E / 7 | pGGZ001 | Chloramphenicol and Spectinomycin |
| E / 8 | pGGZ003 | Chloramphenicol and Spectinomycin |
Kit Plate Map - #1000000036
GreenGate plasmids by type
Plasmid data are from Table 1 of original publication (DOI: 10.1371/journal.pone.0083043.t001)
| Plasmid Category | Name | Insert Type | Overhangs | Bacterial resistance | Addgene ID | Well Location |
|---|---|---|---|---|---|---|
| Empty entry vectors (ccdB+) | pGGA000 | Plant promoter | A-B | Ampicillin | 48856 | D7 |
| pGGB000 | N-tag | B-C | Ampicillin | 48857 | D8 | |
| pGGC000 | CDS | C-D | Ampicillin | 48858 | D9 | |
| pGGD000 | C-tag | D-E | Ampicillin | 48859 | D10 | |
| pGGE000 | Plant terminator | E-F | Ampicillin | 48860 | D11 | |
| pGGF000 | Plant resistance cassette | F-G | Ampicillin | 48861 | D12 | |
| pGGH000 | N-tag + CDS + C-tag + terminator | B-F | Ampicillin | 48862 | E1 | |
| pGGI000 | N-tag + CDS + C-tag | B-E | Ampicillin | 48863 | E2 | |
| Empty intermediate vectors for two expression cassettes on one T-DNA (ccdB+) | pGGM000 | Assembly of expression cassette #1 (without plant resistance) | A-H | Kanamycin | 48864 | E5 |
| pGGN000 | Assembly of expression cassette #2 (with plant resistance) | H-G | Kanamycin | 48865 | E6 | |
| Empty destination vectors (ccdB+) | pGGY001 | Plant resistance at RB | A-G | Gentamicin | 48866 | E4 |
| pGGY003 | Plant resistance at LB | A-G | Gentamicin | 48867 | E3 | |
| pGGZ001 | Plant resistance at RB | A-G | Spectinomycin | 48868 | E7 | |
| pGGZ003 | Plant resistance at LB | A-G | Spectinomycin | 48869 | E8 | |
| Plant promoters | pGGA002 | AP3 (APETALA3; internal BsaI site removed) promoter | A-B | Ampicillin | 48813 | A1 |
| pGGA003 | WUS (WUSCHEL; 4.4 kb) promoter | A-B | Ampicillin | 48814 | A2 | |
| pGGA004 | 35S (Cauliflower mosaic virus 35S; internal BsaI site removed) promoter | A-B | Ampicillin | 48815 | A3 | |
| pGGA006 | UBQ10 (UBIQUITIN10) promoter | A-B | Ampicillin | 48816 | A4 | |
| pGGA008 | ALCA (A. nidulans alc regulon) promoter | A-B | Ampicillin | 48817 | A5 | |
| pGGA012 | RPS5A (RIBOSOMAL PROTEIN 5A) promoter | A-B | Ampicillin | 48818 | A6 | |
| N-tags | pGGB001 | mCherry-linker | B-C | Ampicillin | 48819 | A7 |
| pGGB002 | Ω (Omega- element) | B-C | Ampicillin | 48820 | A8 | |
| pGGB003 | B-dummy (default random sequence if no specific N-tag is desired) | B-C | Ampicillin | 48821 | A9 | |
| pGGB005 | SV40 NLS (SIMIAN VIRUS 40 NUCLEAR LOCALIZATION SIGNAL) | B-C | Ampicillin | 48822 | A10 | |
| pGGB006 | ER signal sequence | B-C | Ampicillin | 48823 | A11 | |
| Coding sequences | pGGC001 | WUS (internal BsaI site removed) | C-D | Ampicillin | 48824 | A12 |
| pGGC011 | ALCR (A. nidulans alc regulon transcriptional regulator) | C-D | Ampicillin | 48825 | B1 | |
| pGGC012 | GFP-NLS (GREEN FLUORESCENT PROTEIN-NUCLEAR LOCALIZATION SIGNAL) | C-D | Ampicillin | 48826 | B2 | |
| pGGC014 | GFP (A206K) | C-D | Ampicillin | 48827 | B3 | |
| pGGC015 | mCherry | C-D | Ampicillin | 48828 | B4 | |
| pGGC024 | BFP (BLUE FLUORESCENT PROTEIN) | C-D | Ampicillin | 48829 | B5 | |
| pGGC025 | 3x GFP | C-D | Ampicillin | 48830 | B6 | |
| pGGC026 | 3x mCherry | C-D | Ampicillin | 48831 | B7 | |
| pGGC051 | GUS (E. coli ß-GLUCURONIDASE) | C-D | Ampicillin | 48832 | B8 | |
| C-tags | pGGD001 | linker-GFP | D-E | Ampicillin | 48833 | B9 |
| pGGD002 | D-dummy (default random sequence with stop codon if no specific C-tag is desired) | D-E | Ampicillin | 48834 | B10 | |
| pGGD003 | Linker-mCherry | D-E | Ampicillin | 48835 | B11 | |
| pGGD005 | Linker-BFP | D-E | Ampicillin | 48853 | D4 | |
| pGGD006 | SV40 NLS | D-E | Ampicillin | 48836 | B12 | |
| pGGD007 | linker-NLS | D-E | Ampicillin | 48837 | C1 | |
| pGGD008 | ER retrieval signal (HDEL) | D-E | Ampicillin | 48838 | C2 | |
| Plant terminators | pGGE001 | RBCS terminator (from pea) | E-F | Ampicillin | 48839 | C3 |
| pGGE002 | WUS terminator | E-F | Ampicillin | 48840 | C4 | |
| pGGE003 | At1g04880 terminator | E-F | Ampicillin | 48854 | D5 | |
| pGGE005 | At1g76110 terminator | E-F | Ampicillin | 48855 | D6 | |
| pGGE009 | UBQ10 terminator | E-F | Ampicillin | 48841 | C5 | |
| Plant resistance cassettes | pGGF001 | pMAS:BastaR:tMAS | F-G | Ampicillin | 48842 | C6 |
| pGGF002 | p35S (internal BsaI site removed):BastaR:t35S | F-G | Ampicillin | 48843 | C7 | |
| pGGF003 | pMAS:D-AlaR:tMAS | F-G | Ampicillin | 48844 | C8 | |
| pGGF004 | p35S (internal BsaI site removed):D-AlaR:t35S | F-G | Ampicillin | 48845 | C9 | |
| pGGF005 | pUBQ10:HygrR:tOCS | F-G | Ampicillin | 48846 | C10 | |
| pGGF007 | pNOS:KanR:tNOS | F-G | Ampicillin | 48847 | C11 | |
| pGGF008 | pNOS:BastaR (chi sequence removed):tNOS | F-G | Ampicillin | 48848 | C12 | |
| pGGF012 | pMAS:SulfR:t35S | F-G | Ampicillin | 48849 | D1 | |
| Adapters | pGGG001 | FH-adapter | F-H | Ampicillin | 48850 | D2 |
| pGGG002 | HA-adapter | H-A | Ampicillin | 48851 | D3 |