Part:BBa_K5133000

T7 promoter (from plasmid pJL1)

Group: GEC-China (iGEM 2024, team number: #5133)

Brief introduction

This basic part is derived from plasmid pJL1 (Addgene: #69496)^[1], including a conserved DNA sequence of T7 promoter as 5'-taatacgactcactatagggaga-3'^[2]. The plasmid pJL1 is commonly used for the in vitro sfGFP expression of cell-free protein synthesis (CFPS). Hence, this part is used for the construction of three composite parts: BBa_K5133004 (sfGFP generator), BBa_K5133006 (Microcin H47 generator), and BBa_K5133008 (Microcin M generator), for CFPS in our project.

Design and characterization

The plasmid design of this biological part is shown as Figure 1, assembled with iGEM standard backbone pSB1C3. Result of Sanger sequencing shows the correct construction of this part (Figure 2).

Figure 1. Design of basic part BBa_K5133000, generated by SnapGene.

Figure 2. Validation the DNA sequence of BBa_K5133000 by Sanger sequencing, generated by SnapGene.

Usages

This part is used for the construction of three composite parts: BBa_K5133004 (sfGFP generator), BBa_K5133006 (Microcin H47 generator), and BBa_K5133008 (Microcin M generator), for CFPS in our project.

DNA sequence (from 5' to 3')

atcccgcgaaattaatacgactcactatagggagaccacaacggtttccctctagaaataattttgtttaacttt

Red font: conserved T7 promoter

References

[1] https://www.addgene.org/69496/

[2] Conrad, T. et al. Maximizing transcription of nucleic acids with efficient T7 promoters. Communications Biology 3, 439 (2020). doi: 10.1038/s42003-020-01167-x

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 51
  
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 51
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 51
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 32