Part:BBa_K2842669

mScarlet reporter with TerL-C intein on the N terminus

This DNA construct encodes a C terminal segment of the AceL-TerL intein fused to the N terminus of a mScarlet reporter protein with a C terminal StrepTag for purification. The AceL-TerL intein acts as a protein ligase that forms a peptide bond between this protein and another protein bound to a complementary split intein, during this process the intein splices itself out of protein and is not present in the end fusion protein. This construct is a modular platform for the creation of split intein fusion proteins through the SapI restriction sites located immediately upstream and downstream of the mScarlet reporter. BBa_K2832680 was designed to be used in conjunction with this construct as it contains the corresponding N terminal intein segment to enable intein trans-splicing.

Figure 1: Expression of IP at varying IPTG concentrations

Initial experiments suggested that 400 uM of IPTG was optimal for expression(data not shown). A plate reader was used to examine the region of IPTG concentrations around 400 uM

Figure 1: BsaI digestions

(1) BsaI digested Intein Passenger BBa_K2842669
(2) BsaI digested RFP inteins BBa_K2842680
(3) BsaI digested GFP inteins BBa_K2842690
*edited to show relevant bands

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Sequence and Features

Assembly Compatibility:

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

  Illegal NotI site found at 941
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1224
  Illegal BsaI.rc site found at 28
  Illegal SapI site found at 1130
  Illegal SapI.rc site found at 419