Part:BBa_K1159015

Membrane-anchored NanoLuc Luciferase in RFC[25] N-Part

This part codes for a membrane-anchored version of NanoLuc Luciferase for the chassis Physcomitrella patens. Please see subparts for for further description. 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
INCOMPATIBLE WITH RFC[21]
Illegal XhoI site found at 83
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 597
Illegal BsaI.rc site found at 1515

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)
AACAATGGAAACC ... CTCTACAAAACCGGT
ORF from nucleotide position 5 to 1591 (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
501

METDTLLLWVLLLWVPGSTGDTGVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKI
FKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILATGSAWSH
PQFEKGENLYFQSGTGPGQPPFPPPPPFTPPPPQTPNGASGENSTGAIAGGVAAGAALLFAAPAIGFAWWRRRRPIEATGGGSGGGSGTGSKGEELFTGV
VPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAE
VKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLSPDNHYLSTQSALSKDP
NEKRDHMVLLEFVTAAGITHGMDELYKTG*

Sequence features: (with their position in the amino acid sequence, see the list of supported features)

	RFC25 scar (shown in bold):	19 to 20, 22 to 23, 194 to 195, 215 to 216, 279 to 280, 289 to 290
	Strep-tag II:	198 to 205
	TEV cleavage site:	207 to 213

Amino acid composition:

Ala (A)	24 (4.5%)
Arg (R)	17 (3.2%)
Asn (N)	24 (4.5%)
Asp (D)	32 (6.0%)

Cys (C)	3 (0.6%)
Gln (Q)	19 (3.6%)
Glu (E)	29 (5.5%)
Gly (G)	66 (12.5%)

His (H)	15 (2.8%)
Ile (I)	33 (6.2%)
Leu (L)	44 (8.3%)
Lys (K)	28 (5.3%)

Met (M)	8 (1.5%)
Phe (F)	26 (4.9%)
Pro (P)	33 (6.2%)
Ser (S)	25 (4.7%)

Thr (T)	38 (7.2%)
Trp (W)	9 (1.7%)
Tyr (Y)	18 (3.4%)
Val (V)	38 (7.2%)

Amino acid counting

	Total number:	529
	Positively charged (Arg+Lys):	45 (8.5%)
	Negatively charged (Asp+Glu):	61 (11.5%)
	Aromatic (Phe+His+Try+Tyr):	68 (12.9%)

Biochemical parameters

	Atomic composition:	C₂₆₁₆H₄₀₀₈N₆₉₀O₇₇₆S₁₁
	Molecular mass [Da]:	57893.4
	Theoretical pI:	5.41
	Extinction coefficient at 280 nm [M^-1 cm^-1]:	76320 / 76508 (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 (0.70)	good (0.72)	good (0.70)	excellent (0.84)	excellent (0.84)	good (0.74)

Alignments (obtained from PredictProtein.org)

	SwissProt:	P42212 (98% identity on 238 AAs), Q9GV45 (90% identity on 169 AAs)
	TrEML:	Q71RY9 (98% identity on 238 AAs), B6F2F5 (97% identity on 241 AAs)
	PDB:	1gfl (98% identity on 229 AAs), 2y0g (98% identity on 226 AAs)

Predictions (obtained from PredictProtein.org)

Subcellular Localization (reliability in brackets)

	Archaea:	secreted (100%)
	Bacteria:	secreted (75%)
	Eukarya:	cytosol (41%)

Gene Ontology (reliability in brackets)

	Molecular Function Ontology:	-
	Biological Process Ontology:	GO:0018298 (44%), GO:0008218 (31%)

Predicted features:

	Disulfid bridges:	-
	Transmembrane helices:	250 to 267 going inwards

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