Part:BBa_K864444

RFP-Linker-SYFP2

This part is a component of the Uppsala University iGEM 2012 modular small RNA screening system, together with the spot42 sRNA BBa_K864440. It can also be used for studing up- or downregulation of genes by other mechanisms.

Use this system to screen for downregulation of your gene by a random small RNA library. PCR amplify your 5’UTR, adding XbaI and EcoRI sites upstream and a BamHI site downstream, and clone into any backbone carrying this part, replacing the RFP cassette. By cloning with EcoRI and BamHI, the cloning will function as a red/white screening system. You have now created a novel BioBrick, that can be used to screen for translational downregulation of your gene of interest.

SYFP2 (BBa_K864100) is situated downstream of the RFP cassette BBa_J04450 and is connected in frame with a 10 aa glycine-serine repeated linker (BBa_J18922), to prevent folding errors in the SYFP2. Before the linker there is a BamHI restriction site (GGATCC), which translates into glycine-serine in E coli.

When screening for working small RNAs, it's recommended to include the first 15 codons of your gene of interest.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1070
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 781
  Illegal AgeI site found at 893
  
• 1000
  COMPATIBLE WITH RFC[1000]