Part:BBa_K1159112

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)

1
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. coli	B. subtilis	S. cerevisiae	A. thaliana	P. patens	Mammals
	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:	-

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