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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 at $65 per plasmid, and will be shipped as bacterial stabs

Add to Cart
$350 USD + shipping

Available to academics and nonprofits only.

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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

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

Illustration of GreenGate vector design

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.

Illustration of GreenGate entry and destination vectors

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:

Icon GreenGate cloning Kit (264.4 KB)

Resources

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

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 links to its kit plate map on the right. 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 Gentamycin
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 Gentamycin
E / 4 pGGY001
Chloramphenicol and Gentamycin
E / 5 pGGM000
Chloramphenicol and Kanamycin
E / 6 pGGN000
Chloramphenicol and Kanamycin
E / 7 pGGZ001
Chloramphenicol and Spectinomycin
E / 8 pGGZ003
Chloramphenicol and Spectinomycin
Data cached @ 2017-05-22

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
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