4G-Cloning Plasmid Kit
(Kit #
1000000252
)
Depositing Lab: Stephan Gruber
The 4G (Golden-Gate-guided Gibson) cloning kit provides vectors containing elements (promoters, terminators, tags, etc.) for rapid assembly of expression vectors for multi-subunit protein complexes in various expression hosts. Expression cassettes for individual subunits are first assembled by Golden Gate cloning, and then directly combined by Gibson Assembly into acceptor vectors for the desired host.
This kit will be sent as bacterial glycerol stocks in 96-well plate format.
Original Publication
4G cloning: rapid gene assembly for expression of multisubunit protein complexes in diverse hosts. Taschner M, Dickinson JB, Roisné-Hamelin F, Gruber S. Life Sci Alliance. 2024 Dec 2;8(1):e202402899. doi: 10.26508/lsa.202402899. PubMed (Link opens in a new window) Article (Link opens in a new window)
Description
Recombinant expression and purification of multi-subunit protein complexes often requires co-expression of several subunits in the same host cell. The production of expression vectors for such assemblies is a time-consuming process, especially if several versions (e.g. with different affinity tags or tag combinations) need to be screened. The ‘4G’ (Golden-Gate-guided Gibson) cloning kit streamlines this process by providing essential ‘elements’ of expression cassettes (collections of promoters, terminators, tags, and Gibson homology sequences).
Users generate vectors containing the ‘ORF’ elements (the coding sequences of each subunit), and can then directly produce expression cassettes by Golden Gate cloning using the Type IIS restriction enzyme BsaI. The Gibson homology sequences added to the ends of individual cassettes allow for their direct insertion into dedicated acceptor vectors used for protein expression. This streamlined process enables rapid, parallel production of multiple vector variants (e.g. for tag screening) within a single day.
The kit provides acceptor vectors for protein expression in bacteria (E. coli), but the system can also be used for eukaryotic hosts (insect or mammalian cells) together with vectors from the biGBac kit (Addgene #1000000088).
- Figure 1: Overview of the “Golden Gate–guided Gibson” (4G cloning) procedure. In the first step, individual linear gene expression cassettes (GECs) are assembled from individual smaller elements by Golden Gate cloning. These elements are excised from individual circular donor vectors (“pD”; see generic representation next to the element library box), giving specific sticky ends depending on the element type (numbers in white circles). Multiple GECs with compatible homology ends from such reactions are then directly inserted into dedicated linearized acceptor vectors by Gibson assembly without further isolation/purification. As a result, a multi-GEC expression plasmid can be created in a single day from prepared donors. Image reused from Taschner et al. 2024, under Creative Commons Attribution License (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 4G-Cloning Plasmid Kit was a gift from Stephan Gruber (Addgene kit #1000000252)."
For your Reference section:
4G cloning: rapid gene assembly for expression of multisubunit protein complexes in diverse hosts. Taschner M, Dickinson JB, Roisné-Hamelin F, Gruber S. Life Sci Alliance. 2024 Dec 2;8(1):e202402899. doi: 10.26508/lsa.202402899. PubMed (Link opens in a new window) Article (Link opens in a new window)
4G-Cloning Plasmid Kit - #1000000252
- 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
| Chloramphenicol | |
| Ampicillin | |
| Kanamycin | |
| Streptomycin |
Inventory
| Well | Plasmid | Resistance |
|---|---|---|
| A / 1 | pD(P+T)_T7 | Chloramphenicol |
| A / 2 | pD(P+T)_T7N | Chloramphenicol |
| A / 3 | pD(P+T)_T7C | Chloramphenicol |
| A / 4 | pD(P+T)_ph | Chloramphenicol |
| A / 5 | pD(P+T)_phN | Chloramphenicol |
| A / 6 | pD(P+T)_phC | Chloramphenicol |
| A / 7 | pD(P+T)_CMV | Chloramphenicol |
| A / 8 | pD(P+T)_CMVN | Chloramphenicol |
| A / 9 | pD(P+T)_CMVC | Chloramphenicol |
| A / 10 | pD(Gib)_5'alpha | Chloramphenicol |
| A / 11 | pD(Gib)_5'beta | Chloramphenicol |
| A / 12 | pD(Gib)_5'gamma | Chloramphenicol |
| B / 1 | pD(Gib)_5'delta | Chloramphenicol |
| B / 2 | pD(Gib)_3'beta | Chloramphenicol |
| B / 3 | pD(Gib)_3'gamma | Chloramphenicol |
| B / 4 | pD(Gib)_3'delta | Chloramphenicol |
| B / 5 | pD(Gib)_3'omega | Chloramphenicol |
| B / 6 | pD(Nt)_His6 | Chloramphenicol |
| B / 7 | pD(Nt)_His6-TEV | Chloramphenicol |
| B / 8 | pD(Nt)_His6-3C | Chloramphenicol |
| B / 9 | pD(Nt)_His6-FLAG3 | Chloramphenicol |
| B / 10 | pD(Nt)_His6-FLAG3-TEV | Chloramphenicol |
| B / 11 | pD(Nt)_His6-HA2-TEV | Chloramphenicol |
| B / 12 | pD(Nt)_His6-MBP-TEV | Chloramphenicol |
| C / 1 | pD(Nt)_His6-GST-TEV | Chloramphenicol |
| C / 2 | pD(Nt)_His6-SUMO | Chloramphenicol |
| C / 3 | pD(Nt)_GFP | Chloramphenicol |
| C / 4 | pD(Nt)_GFP-TEV | Chloramphenicol |
| C / 5 | pD(Nt)_GFP-3C | Chloramphenicol |
| C / 6 | pD(Nt)_TS-3C | Chloramphenicol |
| C / 7 | pD(Nt)_His10-TS-3C | Chloramphenicol |
| C / 8 | pD(Nt)_HALO | Chloramphenicol |
| C / 9 | pD(Ct)_His8 | Chloramphenicol |
| C / 10 | pD(Ct)_3C-His8 | Chloramphenicol |
| C / 11 | pD(Ct)_3C-GFP | Chloramphenicol |
| C / 12 | pD(Ct)_3C-Venus-His8 | Chloramphenicol |
| D / 1 | pD(Ct)_CPD-His10 | Chloramphenicol |
| D / 2 | pD(Ct)_TS | Chloramphenicol |
| D / 3 | pD(Ct)_3C-TS | Chloramphenicol |
| D / 4 | pD(Ct)_3C-TS-His10 | Chloramphenicol |
| D / 5 | pD(Ct)_Avi-3C-TS | Chloramphenicol |
| D / 6 | pD(Ct)_FLAG | Chloramphenicol |
| D / 7 | pD(Ct)_FLAG3 | Chloramphenicol |
| D / 8 | pD(Ct)_MYC9 | Chloramphenicol |
| D / 9 | pD(Ct)_PK6 | Chloramphenicol |
| D / 10 | pD(Ct)_HALO | Chloramphenicol |
| D / 11 | pMulti_A_ccdB | Ampicillin |
| D / 12 | pMulti_K_ccdB | Kanamycin |
| E / 1 | pMulti_S_ccdB | Streptomycin |