Part:BBa_K4807026

hemA(s) insert

In order to achieve high-level overexpression of hemL (BBa_K4759025), hemA(s) (BBa_K4807000) and hemF (BBa_K2779006) we employed pET-29c(+), (BBa_K4807019) a high-copy-number plasmid, as the vector for these three genes.

pET-29c(+) features a ColE1/pMB1/pBR322/pUC origin of replication and it is kanamycin resistant.

This vector already includes one T7 promoter, one RBS and one t7 terminator, so in order to regulate the second unit of our insert, we integrated a T7 promoter+RBS sequence (BBa_K4807001), a second RBS sequence and a T7 terminator (BBa_K4807002), all originated from pETDuet-1.

To ensure their proper expression, each gene had to include a tag for its detection using Western blotting. Consequently, we incorporated an HA-tag (BBa_K1150016) and a Myc-tag (BBa_K3071017) in our hemA(s) and hemF inserts respectively. The hemL gene utilizes the S-tag provided by the vector.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 236
  Illegal PstI site found at 993
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 236
  Illegal PstI site found at 993
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 236
  Illegal PstI site found at 993
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 236
  Illegal PstI site found at 993
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1070
  Illegal SapI.rc site found at 221