Part:BBa_K3425020

iGEM Type IIS standard terminator template

This series of iGEM Type IIS templates present easy to use blueprints to design parts for this assembly standard. These parts provide ready-made sequences which contain the different fusion sites for a promoter, RBS, CDS and terminator part according to the following image*.

By adding the fusion and restriction sites by DNA synthesis or PCR, the part is ready to be cloned into the Type IIS universal acceptor pSB1C00. More information about this backbone can be found in its page. It can also be found, together with all our experience in this cloning method, in Team Uppsala 2020's iGEM Type IIS standard guidebook.

*Note: This image was taken from the pSB1C00 page to provide visual aid for the explanation without redirecting to another page.

This part is part of iGEM Uppsala 2020's iGEM Type IIS standard collection.

Usage and biology

This part is a terminator template, and it is used to clone terminator-type parts (such as BBa_B0015) into pSB1C00, the first step towards assembling transcriptional units with iGEM Type IIS assembly. The template is flanked by fusion sites FS_e (GCTT) and FS_f (CGCT), which will allow it to take the fourth and last position when using it for assembly to a Level 1 or pOdd plasmid (such as pSB1K01).

Follow these steps to use this template:

Obtain the template sequence from "Sequence and features"
Substitute the A homopolymer (annotated as terminator template) by the desired terminator part
Order the part*.

For ease of use, we provide a .dna file which also contains example primers. This file can be found in Team Uppsala 2020's Parts page.

*Note: Terminators are short parts and, going by length, should be ordered as oligos and extended by PCR [1]. However, they tend to be repetitive and form secondary structures, so it is highly likely that this strategy would not work as the primers could anneal in many different ways.

We recommend using BBa_B0015 since it is present in the DNA Distribution Kit already in pSB1C00 [2]. Alternatively, it could be better to order it as dsDNA.

The following table details the part numbers and names of the design templates.

Registry Number	Name
BBa_K3425017	Promoter template
BBa_K3425018	RBS template
BBa_K3425019	CDS template
BBa_K3425020	Terminator template

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal XhoI site found at 32
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI site found at 5
Illegal SapI.rc site found at 39

Note: The part contains restriction sites for its cloning into Level 0 universal Type IIS acceptor pSB1C00, therefore it is marked as not compatible with RFC1000 standard, even though it is.

References

[1] https://parts.igem.org/Help:Promoters/Construction#Constructing_a_part_from_synthetic_oligos

[2] 2019 Distribution Kit Plate 6, 12F. https://parts.igem.org/assembly/plates.cgi?id=6370