Part:BBa_K1159207

Membrane-anchored SpyTag in RFC[25] N-Part

This part codes for a membrane-anchored version of SpyTag BBa_K1159201 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
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 159
Illegal BsaI.rc site found at 1077

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 ... CTCTACAAAACCGGT
ORF from nucleotide position 5 to 1153 (excluding stop-codon)

Amino acid sequence: (RFC 25 scars in shown in bold, other sequence features underlined; both given below)

1
101
201
301

MPGEVAWWRPLFLIALMPIGVLSNAEGDALNTTGAHIVMVDAYKPTKTGSAWSHPQFEKGENLYFQSGTGPGQPPFPPPPPFTPPPPQTPNGASGENSTG
AIAGGVAAGAALLFAAPAIGFAWWRRRRPIEATGGGSGGGSGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPW
PTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQ
KNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLSPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYKTG*

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

	RFC25 scar (shown in bold):	33 to 34, 48 to 49, 69 to 70, 133 to 134, 143 to 144
	Spy-tag:	35 to 47
	Strep-tag II:	52 to 59
	TEV cleavage site:	61 to 67

Amino acid composition:

Ala (A)	27 (7.0%)
Arg (R)	11 (2.9%)
Asn (N)	18 (4.7%)
Asp (D)	20 (5.2%)

Cys (C)	2 (0.5%)
Gln (Q)	12 (3.1%)
Glu (E)	22 (5.7%)
Gly (G)	47 (12.3%)

His (H)	12 (3.1%)
Ile (I)	18 (4.7%)
Leu (L)	27 (7.0%)
Lys (K)	23 (6.0%)

Met (M)	8 (2.1%)
Phe (F)	19 (5.0%)
Pro (P)	30 (7.8%)
Ser (S)	19 (5.0%)

Thr (T)	27 (7.0%)
Trp (W)	6 (1.6%)
Tyr (Y)	13 (3.4%)
Val (V)	22 (5.7%)

Amino acid counting

	Total number:	383
	Positively charged (Arg+Lys):	34 (8.9%)
	Negatively charged (Asp+Glu):	42 (11.0%)
	Aromatic (Phe+His+Try+Tyr):	50 (13.1%)

Biochemical parameters

	Atomic composition:	C₁₈₆₈H₂₈₄₅N₄₉₉O₅₅₇S₁₀
	Molecular mass [Da]:	41525.7
	Theoretical pI:	5.85
	Extinction coefficient at 280 nm [M^-1 cm^-1]:	52370 / 52495 (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 (0.73)	good (0.75)	good (0.76)	excellent (0.85)	excellent (0.81)	good (0.71)

Alignments (obtained from PredictProtein.org)

	SwissProt:	P42212 (98% identity on 237 AAs), Q9U6Y3 (25% identity on 217 AAs)
	TrEML:	B6UPG7 (98% identity on 237 AAs), Q71RY9 (98% identity on 237 AAs), Q8GHE2 (98% identity on 237 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 (68%)
	Eukarya:	cytosol (32%)

Gene Ontology (reliability in brackets)

	Molecular Function Ontology:	-
	Biological Process Ontology:	GO:0018298 (45%), GO:0008218 (32%)

Predicted features:

	Disulfid bridges:	-
	Transmembrane helices:	104 to 121 going inwards

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