Golden GATEway Cloning Kit
(Kit #
1000000054
)
Depositing Lab: Joachim Wittbrodt
The Golden GATEway cloning system combines Golden Gate and Multisite Gateway cloning for construction of complex plasmids in a predefined order. This system was specifically designed for generating transgenesis constructs, but is also suitable for creating fusion proteins, and can be used in many different model organisms.
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
Golden GATEway cloning--a combinatorial approach to generate fusion and recombination constructs. Kirchmaier S, Lust K, Wittbrodt J. PLoS One 2013 Oct 7;8(10):e76117. doi: 10.1371/journal.pone.0076117. PubMed PMID 24116091.
Description
The Golden GATEway cloning system is a combinatorial assembly method that combines Golden Gate (Type IIs endonucleases) cloning with Multisite Gateway (recombination) cloning to construct complex plasmids in an efficient and modular way. Pre-designed fragments can be cloned in a desired order and combined into a final expression vector, while retaining the ability to reuse the predefined fragments in order to efficiently add, remove or replace one or more of the specific fragments. This system was specifically designed for generating transgenesis constructs, but is also suitable for creating fusion proteins. Since the final destination vector determines the use of the plasmid, the system can be used in many different model organisms.
Golden GATEway Method Overview
Figure from Kirchmaier et al., 2013. doi:10.1371/journal.pone.0076117.g001
In preparation for using the Golden GATEway cloning system, desired DNA sequences must be cloned into the Golden Gate entry vectors ( pGGEV ) either by TA cloning or by restriction enzyme cloning using BamHI and KpnI. The cloned fragment in the entry vector is flanked by BsaI sites used for Golden Gate assembly and can be subcloned into other entry vectors, depending on the desired order of fragments in the final expression vector. Once these predefined blocks are constructed, the first cloning step utilizes Golden Gate cloning to assemble the entry vectors sequentially into an appropriate Gateway vector ( p5E_GGDest, pME_GGDest or p3E_GGDest ). The second cloning step uses these Multisite Gateway-compatible vectors to generate the final expression vector in an LR recombination reaction.
Kit Documentation
Golden GATEway Cloning Kit.pdf 120.4Â KBResources
For updated protocols and guidelines on using the Golden GATEway plasmids, please visit Golden GATEway cloning by Stephan Kirchmaier.
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 Golden GATEway plasmid kit was a gift from Joachim Wittbrodt (Addgene kit # 1000000054)"
For your Reference section:
Golden GATEway cloning--a combinatorial approach to generate fusion and recombination constructs. Kirchmaier S, Lust K, Wittbrodt J. PLoS One 2013 Oct 7;8(10):e76117. doi: 10.1371/journal.pone.0076117. doi: 10.1371/journal.pone.0083043. PubMed PMID 24116091
Golden GATEway Cloning Kit - #1000000054
- 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 |
Inventory
| Well | Plasmid | Resistance |
|---|---|---|
| A / 1 | pGGEV_+1_Linker | Ampicillin |
| A / 2 | pGGEV_-1_Linker | Ampicillin |
| A / 3 | pGGEV_2_Linker | Ampicillin |
| A / 4 | pGGEV_3_Linker | Ampicillin |
| A / 5 | pGGEV_4_Linker | Ampicillin |
| A / 6 | pGGEV_5_Linker | Ampicillin |
| A / 7 | pGGEV_6_Linker | Ampicillin |
| A / 8 | pGGEV_7_Linker | Ampicillin |
| A / 9 | pGGEV_2'_Linker | Ampicillin |
| A / 10 | pGGEV_3'_Linker | Ampicillin |
| A / 11 | pGGEV_4'_Linker | Ampicillin |
| A / 12 | pGGEV_5'_Linker | Ampicillin |
| B / 1 | pGGEV_6'_Linker | Ampicillin |
| B / 2 | pGGEV_7'_Linker | Ampicillin |
| B / 3 | pGGEV_8'_Linker | Ampicillin |
| B / 4 | pGGEV_+1_XcmI-LacZ | Ampicillin |
| B / 5 | pGGEV_-1_XcmI-LacZ | Ampicillin |
| B / 6 | pGGEV_2_XcmI-LacZ | Ampicillin |
| B / 7 | pGGEV_3_XcmI-LacZ | Ampicillin |
| B / 8 | pGGEV_4_XcmI-LacZ | Ampicillin |
| B / 9 | pGGEV_5_XcmI-LacZ | Ampicillin |
| B / 10 | pGGEV_6_XcmI-LacZ | Ampicillin |
| B / 11 | pGGEV_7_XcmI-LacZ | Ampicillin |
| B / 12 | pGGEV_2'_XcmI-LacZ | Ampicillin |
| C / 1 | pGGEV_3'_XcmI-LacZ | Ampicillin |
| C / 2 | pGGEV_4'_XcmI-LacZ | Ampicillin |
| C / 3 | pGGEV_5'_XcmI-LacZ | Ampicillin |
| C / 4 | pGGEV_6'_XcmI-LacZ | Ampicillin |
| C / 5 | pGGEV_7'_XcmI-LacZ | Ampicillin |
| C / 6 | pGGEV_8'_XcmI-LacZ | Ampicillin |
| C / 7 | pGGEV_-1_FRT_+1_OA- | Ampicillin |
| C / 8 | pGGEV_3_FRT_+1_OA- | Ampicillin |
| C / 9 | pGGEV_2_+nls-eGFP-HA-_+1_BK+ | Ampicillin |
| C / 10 | pGGEV_4'_+nls-mCherry-Flag-_+1_BK+ | Ampicillin |
| C / 11 | pGGW_SM_BglII | Kanamycin |
| C / 12 | pGGW_SM_BamHI | Kanamycin |
| D / 1 | p3E_GGWDest- | Kanamycin |
| D / 2 | p3E_GGWDest+ | Kanamycin |
| D / 3 | p5E_GGWDest- | Kanamycin |
| D / 4 | p5E_GGWDest+ | Kanamycin |
| D / 5 | pME_GGWDest- | Kanamycin |
| D / 6 | pME_GGWDest+ | Kanamycin |
| D / 7 | pGGDestSC-ATG | Kanamycin |
| D / 8 | pGGDestSC+ATG | Kanamycin |