Part:BBa_K3425017

iGEM Type IIS standard promoter template

This series of iGEM Type IIS templates present easy to use blueprints to design parts for this assembly methods. 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.

Usage and biology

This part is a promoter template, and it is used to clone promoter-type parts (such as BBa_J23119) into pSB1C00, the first step towards assembling transcriptional units with iGEM Type IIS assembly. The template is flanked by fusion sites FS_a (GGAG) and FS_b (TACT), which will allow it to take the first position when using it for assembly to a Level 1 or pOdd plasmid.

Follow these steps to use this template:

1. Obtain the template sequence from "Sequence and features"
2. Substitute the A homopolymer (annotated as Promoter template) by the desired promoter part
3. Order the corresponding primers to anneal and extend*.

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: These parts are short so they should be ordered as oligos and extended by PCR [1]. The Fw and Rv oligos should overlap by at least 20bp. If the entire part (including fusion sites and SapI sites) is less than 60bp long, each strand can be ordered as an oligo and then annealed directly.

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

Registry Number	Name	Mimics
BBa_K3425023	pSB1K01-DY	TU1
BBa_K3425024	pSB1K02-DY	TU2
BBa_K3425025	pSB1K03-DY	TU3
BBa_K3425026	pSB1K04-DY	TU4

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
  

References

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