The plasmid universal entry vector generator (pUEG) is a plasmid that can be used to generate any GreenGate entry vector (e.g. "A" or "F"). This is done by combining pUEG with a purified PCR product of your module and doing an assembly (GreenGate) reaction with type IIS restriction enzyme PaqCI/AarI. Insertion into pUEG utilises separate non-GreenGate overhangs while your desired GreenGate overhangs are specified on the primer extension. The reaction is efficient and bacterial selection can be done by counter-selecting purple colonies (Piepers et al., 2023).
Settings
Settings
Primer optimimisation
Silent mutation
PaqCI domestication
Annealing length
21
Advanced settings
Advanced settings
C/G trimming of flanking primers
Add overhangs
5'
3'
Add illegal sites
5'
3'
Change extension [flanking primers]
FW:
5'
3'
RV:
5'
3'
Change extension [site mutations]
FWRV
:
5'
3'
Recognition and cut site spacing
References
References
If you use GreenGate 1.0 in your scientific work, please cite:
Lampropoulos A, Sutikovic Z, Wenzl C, Maegele I, Lohmann JU, et al. (2013) GreenGate - A Novel, Versatile,
and Efficient Cloning System for Plant Transgenesis. PLOS ONE 8(12): e83043. https://doi.org/10.1371/journal.pone.0083043
If you use GreenGate 2.0 in your scientific work, please cite:
Piepers, M., Erbstein, K., Reyes-Hernandez, J., Song, C., Tessi, T., Petrasiunaite, V., Faerber, N., Distel,
K.,
and Maizel, A. (2023). GreenGate 2.0: Backwards compatible addons for assembly of complex transcriptional
units
and their stacking with GreenGate. PLoS One 18, e0290097. https://doi.org/10.1371/journal.pone.0290097
Changelog
Changelog
2025-03-13
- Primer optimisation disabled.
- Single annealing length slider for all primers and primer pairs.
- Incase identical domestication overhangs are generated, new domestication primers are generated until all overhangs are unique.
2025-03-14
- Removed manual domestication because redundancy.
- Added overhangs to fragments.
- Adjusted annoying mouseover behaviour of magic fragments.
2025-03-15
- Primer optimisation fixed. The correct basepairs are now added.
- Removed redundant primer boxes.
- Added primer pair score (magic score) that indicates how well the primer pair will work, theorectically.
- Added a feature to split fragments > 1500 bps. With support for GreenGate 1.0.
- Added notifications if overhangs between fragments are identical.
- Added the posibility to download pGGX if using GreenGate 1.0, and updated annotations of both pUEG and pGGX.
2025-03-18
- Added the function to add multiple mutagenesis or domain removals by clicking the magic button.
- More information on primer pairs, and exact information of bp mutations and fragment length (including extensions).
- Many code modifications in the background to allow merging of fragments in the future. So double check primer sequences.
2025-03-19
- Reorganisation and removal of elements to make the tool more intuitive, hopefully.
- Added a checklist with instructions.
- Mutations are now annotated in the downloadable GenBank file.
2025-03-20
- Reorganisation of buttons, warnings, and graphical elements to optimise user-friendliness and minimise clutter.
2025-03-21
- Added a function to merge small fragments with nearest junction.
2025-03-22
- Further improvements of user-friendliness by removing visual clutter, reorganisation of buttons, adding headers, and adding new buttons for domain removal and mutagenesis.
2025-03-23
- Minor bug fixes
Entry vector generator
Primers for digestion-ligation or GoldenGate-like reaction
Primer optimisation
Entry vector map
DNA migration pattern
Checklist
Follow this checklist if you want detailed instructions
1. Select your cloning method. GreenGate 1.0 for pGGX, and GreenGate 2.0 for pUEG. GreenGate 2.0 is the default setting.
2. Insert your raw module sequence (without vector backbone and mutations). For domestication, it is important that you enter it starting from the first codon so that silent mutations can be introduced.
3. Select your GreenGate overhangs, and if you are constructing a coding sequence, click on the frameshift button to correct the open reading frame.
Optional. Change the settings in the top left. The default settings are optimised for regular use. Additionaly, you can use the optional mutation tools for domain removal or mutagenesis. Switch between the instructions at the top to find instructions for these tools.
4. Press the magic button to domesticate and apply any mutations from domain removal or site-directed mutagenesis. A new window with generated primers and fragments will appear. Primers are paired to generate the shown fragments.
5. Check your fragments. Error messages will appear if something is wrong, for example if you have identical overhangs between fragments. If there are no error messages, then you do not have to do anything. Furthermore, you can split large fragments (> 1.5kb) which may make cloning easier, or merge small fragments (< 100bp) by including mutations for two sites on a single primer.
6. Copy the primers and order them. Each primer pair is optimised for PCR. The magic score indicates how well your primer pair is optimised. Click the info icon for more details, colored values are problematic. But don't worry, unoptimised primers usually work for PCR. You can adjust the annealing length in the settings to attempt to get a better score. As this tool is quite new, it is wise to do a double check on one of the primers to see if the sequence is correct. Lately I've been testing primer sequences and I am confident that regular primers and domestication primers are correct.
7. Download a virtual vector map with your (mutated) module. Change the filename by altering the text.
Optional. Write down the annealing temperature (Ta) and download the virtual gel, which will help you once you have your primers and start cloning.
8. If something went wrong, then it is likely that you encountered a warning. If you encounter any problems, a feature is unintuitive, or you want a new function, then use the feedback button on the top of this page to inform me.
1. Select your cloning method
2. Paste a module sequence
(Length: 0bp)
& 3. select your overhangs
1st codon
5'
3'
4. Press the magic button
Select overhangs
Enter a valid module sequence.
Enter a module sequence that is at least 50bp.
There are two different mutations at the same location.
A mutation was found at a basepair that does not exist in the module sequence.
Mismatch between logged mutation and index of module sequence.
5. Check your fragments
Consider disabling silent mutation
Are you sure you want an "A" overhang on the reverse flank?
Are you sure you want a "G" overhang on the forward flank?
You have identical GreenGate overhangs on both flanks.
Select GreenGate overhangs on both sides.
A start or stop codon was mutated. The last basepair was changed to an *. Take a look at your primer sequences and do a manual edit.
There is a small fragment. You can merge it with the nearest junction and include mutations for both sites on a single primer.
+ Eases PCR fragment purification
- Increases basepair ambiguity
- Can increase annealing temperature
There is a large fragment. You may want to split this fragment in two
+ Eases PCR amplification
- More fragments to amplify
5'
3'
6. Copy your primers
7. Download your entry vector
Optional: Download the DNA migration pattern
Maximum number of lanes (12) is reached.
Optional mutations
Forward flanking primer
Forward flanking primer
5'
3'
Reverse flanking primer
Reverse flanking primer
5'
3'
Domain removal
Domain removal
Unwanted domain sequence
5'
3'
Select which site should be removed
Apply mutation(s)
Mutation(s) log
The removal domain has to be away from the flanks.
Enter a domain sequence.
This domain was already removed.
Domain was not found in the module sequence.
Site directed mutagenesis
Site directed mutagenesis
Notifcation: Currently this tool can only be used to mutate one or multiple individual codons at distinct locations ( > 100bp apart).
Enter a target site for mutation
Target site
5'
3'
The target sequence was not found in the module sequence.
The target sequence was found more than once, increase the length of the target site.
