Part:BBa_K1159111

Membrane-associated Thermonuclease NucA with TEV site and SV40 NLS in RFC[25] 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[25] N-part prefix and suffix. Note: This means only protein fusions to the C-terminus of this part is possible, adding promoters (typically RFC[10]) into the prefix or terminators/IRES (typically RFC[10]) into the suffix of this part is nevertheless possible.

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
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Protein data table for BioBrick BBa_ automatically created by the BioBrick-AutoAnnotator version 1.0
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) 1  101  201  301  MPGEVAWWRPLFLIALMPIGVLSNAEGDALNTTGPGQPPFPPPPPFTPPPPQTPNGASGENSTGAIAGGVAAGAALLFAAPAIGFAWWRRRRPIEATGGG GGSGGGGSGGGGSGGGGSGGGGSGENLYFQSGTGAELIPEPPKKKRKVELGTATGATSTKKLHKEPATLIKAIDGDTVKLMYKGQPMTFRLLLVDTPETK HPKKGVEKYGPEASAFTKKMVENAKKIEVEFDKGQRTDKYGRGLAYIYADGKMVNEALVRQGLAKVAYVYKPNNTHEQHLRKSEAQAKKEKLNIWSEDNA DSGQTG*
Sequence features: (with their position in the amino acid sequence, see the list of supported features) RFC25 scar (shown in bold):  33 to 34, 97 to 98, 133 to 134, 154 to 155 TEV cleavage site:  125 to 131 SV40 nuclear localization sequence:  142 to 148
Amino acid composition: Ala (A) 32 (10.5%) Arg (R) 11 (3.6%) Asn (N) 11 (3.6%) Asp (D) 9 (2.9%) Cys (C) 0 (0.0%) Gln (Q) 9 (2.9%) Glu (E) 20 (6.5%) Gly (G) 49 (16.0%) His (H) 4 (1.3%) Ile (I) 11 (3.6%) Leu (L) 21 (6.9%) Lys (K) 27 (8.8%) Met (M) 6 (2.0%) Phe (F) 9 (2.9%) Pro (P) 27 (8.8%) Ser (S) 14 (4.6%) Thr (T) 20 (6.5%) Trp (W) 5 (1.6%) Tyr (Y) 8 (2.6%) Val (V) 13 (4.2%)
Amino acid counting Total number: 306 Positively charged (Arg+Lys): 38 (12.4%) Negatively charged (Asp+Glu): 29 (9.5%) Aromatic (Phe+His+Try+Tyr): 26 (8.5%)	Biochemical parameters Atomic composition: C1423H2241N399O427S6 Molecular mass [Da]: 31963.2 Theoretical pI: 9.47 Extinction coefficient at 280 nm [M-1 cm-1]: 39420 / 39420 (all Cys red/ox)
Plot for hydrophobicity, charge, predicted secondary structure, solvent accessability, transmembrane helices and disulfid bridges
Codon usage Organism: E. coli B. subtilis S. cerevisiae A. thaliana P. patens Mammals Codon quality (CAI): good (0.75) good (0.78) good (0.76) excellent (0.82) good (0.79) good (0.67)
Alignments (obtained from PredictProtein.org) SwissProt: P00644 (98% identity on 153 AAs), Q5HHM4 (97% identity on 153 AAs) TrEML: A3QSR2 (97% identity on 143 AAs), A3QSR3 (97% identity on 143 AAs) PDB: 1ey0 (100% identity on 136 AAs), 1eyd (100% identity on 136 AAs), 1nsn (100% identity on 138 AAs), 1snc (100% identity on 134 AAs), 1sta (100% identity on 134 AAs), 1stb (100% identity on 136 AAs), 1stg (100% identity on 136 AAs), 1sth (100% identity on 136 AAs), 1stn (100% identity on 136 AAs), 1sty (100% identity on 136 AAs)
Predictions (obtained from PredictProtein.org)
Subcellular Localization (reliability in brackets) Archaea: cytosol (100%) Bacteria: secreted (94%) Eukarya: cytosol (38%)	Gene Ontology (reliability in brackets) Molecular Function Ontology: - Biological Process Ontology: GO:0030541 (28%)
Predicted features: Disulfid bridges:  - Transmembrane helices: 69 to 86 going inwards
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