Part:BBa_K2086000:Design
napDABC (Periplasmic Nitrate Reductase)
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1991
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 479
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 324
Illegal SapI.rc site found at 238
Illegal SapI.rc site found at 1734
Design Notes
The part had an illegal restriction site in the NapA gene. The NapDA genes and NapBC genes aren't normally adjacent to each other and so were assembled using HiFi assembly.
The following primers were used to PCR fragments of the nap operon from E. coli
NapA(R) - AATGGCTTACACCTTCTCCAGT
NapA (F) - CGCGCCGTTGTTGGTCAGCA
NapBC(F) - TGGAGAAGGTGTAAGCCATTATGAAAAGCCATGACCTGAA
NapBC(R) - tgcagcggccgctactagtattaAAAACCTGGCTCGACTT
The following sequence was ordered from IDT due to necessary removal of restriction enzyme sites that are found in the chromosomal DNA of E. coli and the need to add the BioBrick prefix.
GAATTCGCGGCCGCTTCTAGATGCACACTAACTGGCAAGTTTGCAGCCTGGTCGTGCAGGCCAAAAGCGAACGAATTTCAGACATCAGCACCCAACTGAAC GCCTTTCCCGGCTGTGAAGTTGCTGTCAGCGACGCGCCGAGCGGTCAGTTGATTGTGGTGGTGGAAGCAGAAGACAGCGAAACGCTGATCCAAACCATTGAGT CAGTACGCAACGTAGAGGGCGTGCTGGCGGTGTCGCTGGTTTATCACCAGCAGGAAGAGCAAGGTGAGGAAACACCATGAAACTCAGTCGTCGTAGCTTTATG AAAGCTAACGCCGTTGCGGCGGCTGCGGCGGCT GCCGGTCTCAGCGTGCCGGGCGTTGCCCGCGCCGTTGTTGGTCAGCAGGAAGCCATCAAA
Before HiFi assembly could be completed we realized that the overlap between the fragments was too small in some places and so the following primers were used to lengthen their corresponding fragments and increase the amount of overlap.
NapDA (F) - TTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCT
NapDA (R) - TTTGATGGCTTCCTGCTGACCAACAACGG
NapBC (R) - TTTTTTGCCGGACTGCAGCGGCCGCTA
NapBC(F) - TGGAGAAGGTGTAAGCCATTATGAAAAGCCATGACCTGAA
Source
The napDABC gene sequence contains sections of the nap operon derived from the genomic DNA of E. coli (MG1655).