Coding

Part:BBa_K1159112

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

FRET Reporter for TEV Protease activity (eCFP_TEV-site-linker_SYFP2) in RFC[25]

This part encodes a FRET reporter with the chromophores eCFP and SYFP2 that is seperated by a TEV cleavage site. As a FRET pair, excitation of eCFP leads to a energy transfer from eCFP to SYFP2 (FRET) resulting in a fluorescence of SYFP2. In the presence of TEV Protease cleavage actitivy the FRET pair SYFP2 is cleaved off from eCFP and eCFP is not anymore quenched by SYFP2 resulting in the fluorescence of eCFP. This part is flanked by RFC[25] pre- and suffix for further protein fusions.

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, so ATGGCCGGC and ACCGGT were added (in italics) to the 5' and 3' ends: (underlined part encodes the protein)
 ATGGCCGGCGTGAGCAAG ... CTGTATAAAACCGGT
 ORF from nucleotide position -8 to 1473 (excluding stop-codon)
Amino acid sequence: (RFC 25 scars in shown in bold, other sequence features underlined; both given below)

101 
201 
301 
401 
MAGVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERT
IFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIKANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPD
NHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKTGGENLYFQSGTGVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKL
ICTTGKLPVPWPTLVTTLGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYN
SHNVYITADKQKNGIKANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKTG*
Sequence features: (with their position in the amino acid sequence, see the list of supported features)
RFC25 scar (shown in bold): 53 to 54, 242 to 243, 253 to 254
TEV cleavage site: 245 to 251
Amino acid composition:
Ala (A)19 (3.8%)
Arg (R)12 (2.4%)
Asn (N)26 (5.3%)
Asp (D)36 (7.3%)
Cys (C)4 (0.8%)
Gln (Q)17 (3.4%)
Glu (E)33 (6.7%)
Gly (G)52 (10.5%)
His (H)18 (3.6%)
Ile (I)25 (5.1%)
Leu (L)44 (8.9%)
Lys (K)41 (8.3%)
Met (M)9 (1.8%)
Phe (F)24 (4.9%)
Pro (P)20 (4.0%)
Ser (S)19 (3.8%)
Thr (T)35 (7.1%)
Trp (W)3 (0.6%)
Tyr (Y)23 (4.7%)
Val (V)34 (6.9%)
Amino acid counting
Total number:494
Positively charged (Arg+Lys):53 (10.7%)
Negatively charged (Asp+Glu):69 (14.0%)
Aromatic (Phe+His+Try+Tyr):68 (13.8%)
Biochemical parameters
Atomic composition:C2479H3817N653O753S13
Molecular mass [Da]:55233.3
Theoretical pI:5.59
Extinction coefficient at 280 nm [M-1 cm-1]:50770 / 51020 (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 (0.74)good (0.69)acceptable (0.57)good (0.65)excellent (0.85)excellent (0.83)
Alignments (obtained from PredictProtein.org)
SwissProt:P42212 (95% identity on 238 AAs), Q9U6Y3 (26% identity on 217 AAs)
TrEML:B6UPG7 (96% identity on 238 AAs), B7UCZ6 (96% identity on 238 AAs)
PDB:1oxf (98% identity on 225 AAs), 1qxt (98% identity on 226 AAs), 1qy3 (98% identity on 227 AAs), 2awj (98% identity on 227 AAs), 2hfc (98% identity on 230 AAs), 2wsn (98% identity on 225 AAs)
Predictions (obtained from PredictProtein.org)
Subcellular Localization (reliability in brackets)
Archaea:secreted (100%)
Bacteria:cytosol (61%)
Eukarya:cytosol (37%)
Gene Ontology (reliability in brackets)
Molecular Function Ontology: -
Biological Process Ontology:GO:0018298 (44%), GO:0008218 (31%)
 
Predicted features:
Disulfid bridges: -
Transmembrane helices: -
The BioBrick-AutoAnnotator was created by TU-Munich 2013 iGEM team. For more information please see the documentation.
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Categories
Parameters
None