Designed by: Dong-Jiunn Jeffery TRUONG Group: iGEM13_TU-Munich (2013-09-16)
Membrane-associated Thermonuclease NucA with TEV site and SV40 NLS in RFC N-Part
This part encodes a membrane-associated Thermonuclease NucA for the chassis Physcomitrella patens. NucA is a endo/exonuclease that degrades ssDNA/dsDNA/ssRNA/dsRNA. It cannot execute its deadly potential when it is bound to the cytoplasmatic membrane. The region between NucA and the transmembrane domain consists a long linker with a TEV cleavage site and a SV40 nuclear localization signal (NLS). By cleaving the TEV cleavage site with a TEV Protease the nuclease NucA translocates in the nucleus due to its SV40 NLS and degrades the genomic DNA resulting a inevitable induction of programmed cell-death (PCD). This construct is flanked by RFC N-part prefix and suffix. Note: This means only protein fusions to the C-terminus of this part is possible, adding promoters (typically RFC) into the prefix or terminators/IRES (typically RFC) into the suffix of this part is nevertheless possible.
Nucleotide sequence in RFC 25 N-Part using the stop codon in the suffix, so ACCGGT was added (in italics) to the 3' end: (underlined part encodes the protein) AACAATGCCTGGT ... TCAGGTCAAACCGGT ORF from nucleotide position 5 to 922 (excluding stop-codon)
Amino acid sequence: (RFC 25 scars in shown in bold, other sequence features underlined; both given below)