Coding

Part:BBa_K1159207

Designed by: Dong-Jiunn Jeffery TRUONG   Group: iGEM13_TU-Munich   (2013-09-17)

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)

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. coliB. subtilisS. cerevisiaeA. thalianaP. patensMammals
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
The BioBrick-AutoAnnotator was created by TU-Munich 2013 iGEM team. For more information please see the documentation.
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[edit]
Categories
//chassis/eukaryote/ppatens
Parameters
None