Start-Stop Assembly Toolkit
(Kit #
1000000152
)
Depositing Lab: John Heap
Start-Stop Assembly is a multi-part, modular DNA assembly method which is functionally scarless, suitable for combinatorial assembly, and ideal for metabolic engineering in diverse organisms.
This kit will be sent as bacterial glycerol stocks in 96-well plate format. Individual plasmids can be ordered from each plasmid page and will be shipped as bacterial stabs.
Original Publication
Start-Stop Assembly: a functionally scarless DNA assembly framework optimised for metabolic engineering. Taylor GM, Mordaka PM, Heap JT. Nucleic Acids Research. 2019 Feb 20; doi: 10.1093/nar/gky1182. PubMed PMID: 30462270.
Description
Start-Stop Assembly is a multi-part, modular DNA assembly method which is functionally scarless, suitable for combinatorial assembly, and ideal for metabolic engineering in diverse organisms. Start-Stop Assembly provides the same key advantages as other Golden Gate-type modular assembly frameworks (efficient, unbiased, multi-part, hierarchical assembly) but also allows designs to be scarless at CDS boundaries, which are highly-sensitive sites where scars affect mRNA structure, activity of the ribosome binding site, and potentially other functional RNA features. The ‘Start-Stop Assembly Toolkit’ contains the 15 core assembly vectors used to assemble constructs of up to 15 coding sequences. It also contains the 25 promoters, RBSs, terminators and carotenoid pathway coding sequences used for assembly of metabolic pathway-encoding constructs in E. coli (described in Taylor et al. 2019). First-time users may find it helpful to assemble carotenoid pathway libraries as a positive control, because the variety of colored E. coli colonies conveniently show that combinatorial assembly is occurring (see Taylor et al. 2019 linked above for more details).
Schematic overview of Start-Stop Assembly and an example of its implementation to combinatorially assemble a carotenoid pathway.
Kit-Specific Resources
Click below to download the Start-Stop Assembly Quick-Start Guide or Start-Stop Assembly Lab Protocol:
Start-Stop_Assembly_Quick_Start_Guide 57.8Â KB
Start-Stop Assembly Lab Protocol 51.3Â KB
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 Start-Stop Assembly Toolkit was a gift from John Heap (Addgene kit #1000000152).”
For your Reference section:
Start-Stop Assembly: a functionally scarless DNA assembly framework optimised for metabolic engineering. Taylor GM, Mordaka PM, Heap JT. Nucleic Acids Research. (2019) doi: 10.1093/nar/gky1182. PubMed PMID: 30462270.
Start-Stop Assembly Toolkit - #1000000152
- 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 | |
| Tetracycline | |
| Kanamycin | |
| Chloramphenicol |
Inventory
| Well | Plasmid | Resistance |
|---|---|---|
| A / 1 | pStA0::J23100 | Ampicillin |
| A / 2 | pStA0::J23102 | Ampicillin |
| A / 3 | pStA0::J23107 | Ampicillin |
| A / 4 | pStA0::J23113 | Ampicillin |
| A / 5 | pStA0::J23116 | Ampicillin |
| A / 6 | pStA0::J23118 | Ampicillin |
| A / 7 | pStA0::RBSc13 | Ampicillin |
| A / 8 | pStA0::RBSc33 | Ampicillin |
| A / 9 | pStA0::RBSc44 | Ampicillin |
| A / 10 | pStA0::RBSc58 | Ampicillin |
| A / 11 | pSTA0::RBSc36 | Ampicillin |
| A / 12 | pStA0::RBSc42 | Ampicillin |
| B / 1 | pStA0::L3S2P55 | Ampicillin |
| B / 2 | pStA0::L3S2P21 | Ampicillin |
| B / 3 | pStA0::ECK120033737 | Ampicillin |
| B / 4 | pStA0::ECK120019600 | Ampicillin |
| B / 5 | pStA0::crtI | Ampicillin |
| B / 6 | pStA0::crtB | Ampicillin |
| B / 7 | pStA0::crtE | Ampicillin |
| B / 8 | pStA0::dxs | Ampicillin |
| B / 9 | pStA0::crtW | Ampicillin |
| B / 10 | pStA0::crtZ | Ampicillin |
| B / 11 | pStA0::lcyB | Ampicillin |
| B / 12 | pStA0::idi | Ampicillin |
| C / 1 | pStA0::eyfp | Ampicillin |
| C / 2 | pStA0 | Ampicillin |
| C / 3 | pStA1AZ | Tetracycline |
| C / 4 | pStA1AB | Tetracycline |
| C / 5 | pStA1BZ | Tetracycline |
| C / 6 | pStA1BC | Tetracycline |
| C / 7 | pStA1CZ | Tetracycline |
| C / 8 | pStA1CD | Tetracycline |
| C / 9 | pStA1DZ | Tetracycline |
| C / 10 | pStA1DE | Tetracycline |
| C / 11 | pStA1EZ | Tetracycline |
| C / 12 | pStA212 | Kanamycin |
| D / 1 | pStA223 | Kanamycin |
| D / 2 | pStA234 | Kanamycin |
| D / 3 | pStA313 | Chloramphenicol |
| D / 4 | pStA314 | Chloramphenicol |