Part:BBa_K4770018

NR_Nitrate_Reductase_Level_1

Description and Biology

Level 1 vectors are single transcriptional units. This Level 1 contains a constitutive promoter PPSAD, a coding sequence for the Nitrates Reductase in Chlamydomonas reinhardtii linked through F2A (a self-cleaving peptide) to a NanoLuc reporter. With this vector we aim to enhance NR expression, making the first step of the nitrates assimilation pathway constitutive by using the PPSAD promoter and being able to measure this expression through the NanoLuc signal.

How can this part be used

This Level 1 is aimed to later be assembled in a level M in position 5 through MoClos assembly. Should you use this part and need advice on how to use it, please do not hesitate to contact us at algagenix@gmail.com

Source of this part

Original PPSAD sequence: BBa_K4770007 Original NR sequence: BBa_K4770002 Original F2A sequence: BBa_K4770012 Original NanoLuc sequence: BBa_K4770011 Original TPSAD sequence: BBa_K4770008

Design considerations

We performed Chlamydomonas reinhardtii's domestication for NR sequence. This process includes a reduction of the GC% content to make the sequence suitable for commercial synthesis while maintaining it high enough to fit Chlamydomonas reinhardtii's codon usage. This was done using our optimizing software (see AlgaGenix's wiki for additional information). Moreover, recognition sites for BbsI and BsaI were eliminated, changing said codons with synonymous ones. Besides, we also added an intron to perform what is known as intron-mediated enhancement (IME), which studies show aids with stable high-level expression of a foreign gene (Lumbreras et al., 1998) AlgaGenix’s Level 1s are designed to have the same structure and not having to build different pieces with different positions for each gene.

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 1517
  Illegal PstI site found at 2781
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 3361
  Illegal PstI site found at 1517
  Illegal PstI site found at 2781
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 3916
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 1517
  Illegal PstI site found at 2781
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 1517
  Illegal PstI site found at 2781
  Illegal NgoMIV site found at 1406
  Illegal NgoMIV site found at 2000
  Illegal NgoMIV site found at 2615
  Illegal NgoMIV site found at 3732
  Illegal AgeI site found at 1178
  Illegal AgeI site found at 3491
  
• 1000
  COMPATIBLE WITH RFC[1000]