Part:BBa_K2273117:Design

superfold GFP~PenP fusion construct for localization studies

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Design Notes

The penP gene was amplified from the B. subtilis W168 genome sequence using primers with RFC25 restriction site overhangs to enable translational fusions for localisation studies.

Prefix with	EcoRI, NotI, XbaI, RBS, spacer sequence, Start Codon and NgoMIV	GAATTCGCGGCCGCTTCTAGAAGGAGGTGTCAAAATGGCCGGC
Suffix with	AgeI, Stop Codon, SpeI, NotI and PstI	ACCGGTTAAACTAGTAGCGGCCGCTGCAGA

Sites of restriction enzymes generating compatible overhangs are indicated by sharing one color. (EcoRI and PstI are marked in blue, NotI in green, XbaI and SpeI in red, AgeI and NgoMIV in orange)

Primers used for amplification:

Forward Primer containing RFC25 prefix (shown in small letters):

5`-gatcgaattccgcggccgcttctagatCAGAGGAGGCCTGATGgccggcAAGTTGAAAACTAAAGCGTCAATAA-3`

Revers Primer containing RFC25 suffix (shown in small letters):

5`-gatcctgcagcggccgctactagtaTTAaccggtTTTGAGATCGTTAAGGACGAC-3`

Afterwards, the N-Terminus of penP was fused to the C-Terminus of superfold GFP using the RFC25 Standard.

Source

The sequence coding for PenP was derived from the Bacillus subtilis W168 genome sequence.

References

[http://www.subtiwiki.uni-goettingen.de/v3/gene/view/713BAB7190E1F86C55103049B29072F00E0DFFB3]

[http://www.uniprot.org/uniprot/P39824]

Marta Toth et al. (2015) Class D Beta-Lactamases Do Exist in Gram-Positive Bacteria. Nature Chemical Biology (12): 9-14.