Coding

Part:BBa_K1159108

Designed by: Dong-Jiunn Jeffery TRUONG   Group: iGEM13_TU-Munich   (2013-09-16)

N-TEV-Protease_36AALinker_PIF6 (Second part for a light-switchable TEV-Protease) in RFC[25]

This parts encodes a fusion protein consisting the N-terminal half of the TEV Protease as its terminus and first 100 amino acids of PIF6 as its C-terminus. PIF6 interacts with PhyB in the presence of red light. This reaction is reversible by far-red light. Both parts are seperated by a 36 amino acids long flexible linker. This part is flanked by RFC[25] pre- and suffix to allow further protein fusions.

By expressing this part together with its interaction partner BBa_K1159106 that contains the part, which interacts with PIF6, and the C-terminal half of the splitted TEV Protease, they form together a light-switchable TEV Protease system which can be activated by red light and inactivated by far-red light.

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
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 316

Protein data table for BioBrick BBa_ automatically created by the BioBrick-AutoAnnotator version 1.0
Nucleotide sequence in RFC 25, so ATGGCCGGC and ACCGGT were added (in italics) to the 5' and 3' ends: (underlined part encodes the protein)
 ATGGCCGGCGGAGAAAGC ... CATGTTGACACCGGT
 ORF from nucleotide position -8 to 777 (excluding stop-codon)
Amino acid sequence: (RFC 25 scars in shown in bold, other sequence features underlined; both given below)

101 
201 
MAGGESLFKGPRDYNPISSTICHLTNESDGHTTSLYGIGFGPFIITNKHLFRRNNGTLLVQSLHGVFKVKNTTTLQQHLIDGRDMIIIRMPKDFPPFPQK
LKFREPQREERICLVTTNFQTTGGGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSGTGMFLPTDYCCRLSDQEYMELVFENGQILAKGQRSNVSLHN
QRTKSIMDLYEAEYNEDFMKSIIHGGGGAITNLGDTQVVPQSHVAAAHETNMLESNKHVDTG*
Sequence features: (with their position in the amino acid sequence, see the list of supported features)
Factor Xa cleavage site: 80 to 83
RFC25 scar (shown in bold): 122 to 123, 160 to 161
Amino acid composition:
Ala (A)10 (3.8%)
Arg (R)11 (4.2%)
Asn (N)14 (5.3%)
Asp (D)11 (4.2%)
Cys (C)4 (1.5%)
Gln (Q)12 (4.6%)
Glu (E)13 (5.0%)
Gly (G)38 (14.5%)
His (H)10 (3.8%)
Ile (I)15 (5.7%)
Leu (L)19 (7.3%)
Lys (K)11 (4.2%)
Met (M)8 (3.1%)
Phe (F)12 (4.6%)
Pro (P)10 (3.8%)
Ser (S)24 (9.2%)
Thr (T)24 (9.2%)
Trp (W)0 (0.0%)
Tyr (Y)6 (2.3%)
Val (V)10 (3.8%)
Amino acid counting
Total number:262
Positively charged (Arg+Lys):22 (8.4%)
Negatively charged (Asp+Glu):24 (9.2%)
Aromatic (Phe+His+Try+Tyr):28 (10.7%)
Biochemical parameters
Atomic composition:C1209H1902N352O391S12
Molecular mass [Da]:28009.2
Theoretical pI:6.55
Extinction coefficient at 280 nm [M-1 cm-1]:8940 / 9190 (all Cys red/ox)
Plot for hydrophobicity, charge, predicted secondary structure, solvent accessability, transmembrane helices and disulfid bridges 
Codon usage
Organism:E. coliB. subtilisS. cerevisiaeA. thalianaP. patensMammals
Codon quality (CAI):good (0.73)good (0.72)good (0.69)good (0.74)excellent (0.82)good (0.69)
Alignments (obtained from PredictProtein.org)
SwissProt:P04517 (49% identity on 239 AAs), P31999 (36% identity on 222 AAs)
TrEML:Q000U0 (49% identity on 239 AAs), A4UQV2 (48% identity on 239 AAs)
PDB:1lvm (65% identity on 178 AAs), 1q31 (65% identity on 175 AAs)
Predictions (obtained from PredictProtein.org)
Subcellular Localization (reliability in brackets)
Archaea:secreted (100%)
Bacteria:secreted (56%)
Eukarya:cytosol (20%)
Gene Ontology (reliability in brackets)
Molecular Function Ontology:GO:0005515 (70%), GO:0043621 (13%)
Biological Process Ontology:GO:0006508 (32%), GO:0006410 (26%)
 
Predicted features:
Disulfid bridges: -
Transmembrane helices: -
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Categories
Parameters
None