Coding

Part:BBa_K1159015

Designed by: TU Munich 2013   Group: iGEM13_TU-Munich   (2013-05-17)

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_K1159015 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)

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. coliB. subtilisS. cerevisiaeA. thalianaP. patensMammals
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
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