Difference between revisions of "Part:BBa K2404001:Design"

 
(→‎Design Notes)
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===Design Notes===
 
===Design Notes===
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To produce this protein we needed to create a gene construct with the TSH antagonist (with His tags) as the coding sequence. We used the Golden Gate cloning system to do this, and so needed to create upstream restriction enzyme recognition sites (i.e. Type IIS restriction endonucleases) that are suitable for Golden Gate reactions.
 
  
 +
We generated this part through the following steps:
  
 +
 +
- We ordered the following TSH gBlock from IDT
 +
tttGAATTCGCGGCCGCTTCTAGttCGTCTCtCTCAaatgACTGCTCTGTTTCTGATGAGTATGCTGTTCGGACTGGCTTGCGGGCAGGCTATGTCTTTCTGTATCCCCACTGAGTACACCATGCACATCGAGAGGAGAGAATGCGCATACTGTCTGACAATCAACACCACAATTTGCGCCGGGTATTGTATGACTAGGGACATTAATGGCAAGCTGTTCCTGCCAAAATACGCACTGTCCCAGGACGTGTGCACTTACCGGGACTTCATCTACCGCACCGTCGAGATTCCCGGATGTCCTCTGCACGTGGCCCCTTACTTCAGTTATCCAGTCGCTCTGTCATGCAAGTGTGGGAAATGCAACACTGACTATTCTGATTGTATCCATGAAGCTATTAAGACTAACTACTGCACCAAGCCTCAGAAAAGCTATCTGGTGGGCTTTTCCGTCAGCTCCTCTAGTAAGGCCCCCCCTCCATCACTGCCCTCACCTAGCAGACTGCCAGGACCCAGCGACACCCCAATCCTGCCCCAGGCTCCTGACGTGCAGGATTGCCCCGAGTGTACACTGCAGGAAAACCCTTTCTTTAGTCAGCCAGGCGCTCCCATTCTGCAGTGTATGGGATGCTGTTTCTCTCGAGCATACCCTACCCCACTGAGGAGTAAGAAAACAATGCTGGTGCAGAAGGATGTCACATCCGAGTCTACTTGCTGCGTGGCCAAAAGCTATAATCGGGTGACAGTCATGGGCGGATTCAAAGTCGAAGACCACACCGCCTGCCACTGCTCCACCTGCTACTACCATAAAAGTCTGGTTCCGCGTGGATCCCAcCATCAcCAcCATCATTGAgcttCGAGaGAGACGaaTACTAGTAGCGGCCGCTGCAGttt
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- This was introduced into pSB1C3 by digestion with BsmBI. This generated a 5' and 3' BsaI sites for level 1 cloning
  
 
===Source===
 
===Source===

Revision as of 10:44, 6 October 2017


A Thyroid Stimulating Hormone antagonist with His-Tags


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 540
    Illegal PstI site found at 582
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 540
    Illegal PstI site found at 582
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 795
    Illegal XhoI site found at 607
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 540
    Illegal PstI site found at 582
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 540
    Illegal PstI site found at 582
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1
    Illegal BsaI.rc site found at 827


Design Notes

We generated this part through the following steps:


- We ordered the following TSH gBlock from IDT tttGAATTCGCGGCCGCTTCTAGttCGTCTCtCTCAaatgACTGCTCTGTTTCTGATGAGTATGCTGTTCGGACTGGCTTGCGGGCAGGCTATGTCTTTCTGTATCCCCACTGAGTACACCATGCACATCGAGAGGAGAGAATGCGCATACTGTCTGACAATCAACACCACAATTTGCGCCGGGTATTGTATGACTAGGGACATTAATGGCAAGCTGTTCCTGCCAAAATACGCACTGTCCCAGGACGTGTGCACTTACCGGGACTTCATCTACCGCACCGTCGAGATTCCCGGATGTCCTCTGCACGTGGCCCCTTACTTCAGTTATCCAGTCGCTCTGTCATGCAAGTGTGGGAAATGCAACACTGACTATTCTGATTGTATCCATGAAGCTATTAAGACTAACTACTGCACCAAGCCTCAGAAAAGCTATCTGGTGGGCTTTTCCGTCAGCTCCTCTAGTAAGGCCCCCCCTCCATCACTGCCCTCACCTAGCAGACTGCCAGGACCCAGCGACACCCCAATCCTGCCCCAGGCTCCTGACGTGCAGGATTGCCCCGAGTGTACACTGCAGGAAAACCCTTTCTTTAGTCAGCCAGGCGCTCCCATTCTGCAGTGTATGGGATGCTGTTTCTCTCGAGCATACCCTACCCCACTGAGGAGTAAGAAAACAATGCTGGTGCAGAAGGATGTCACATCCGAGTCTACTTGCTGCGTGGCCAAAAGCTATAATCGGGTGACAGTCATGGGCGGATTCAAAGTCGAAGACCACACCGCCTGCCACTGCTCCACCTGCTACTACCATAAAAGTCTGGTTCCGCGTGGATCCCAcCATCAcCAcCATCATTGAgcttCGAGaGAGACGaaTACTAGTAGCGGCCGCTGCAGttt

- This was introduced into pSB1C3 by digestion with BsmBI. This generated a 5' and 3' BsaI sites for level 1 cloning

Source

This modified protein was discovered in a paper by Fares et al in 2001, but without the His-tags. It was ordered as a g-block.

References