Part:BBa_K4807025

hemF 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
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 330
  
• 1000
  COMPATIBLE WITH RFC[1000]