Coding

Part:BBa_K1159016

Designed by: Andreas-David BRUNNER   Group: iGEM13_TU-Munich   (2013-05-17)

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

This part codes for a membrane-anchored version of Laccase from Bacillus pumilus 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 BglII site found at 226
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1644
    Illegal BsaI.rc site found at 2562

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 2638 (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 
601 
701 
801 		MPGEVAWWRPLFLIALMPIGVLSNAEGDALNTTGNLEKFVDELPIPEVAKPVKKNPKQTYYEIAMEEVFLKVHRDLPPTKLWTYNGSLPGPTIHANRNEK
VKVKWMNKLPLKHFLPVDHTIHEGHHDEPEVKTVVHLHGGVTPASSDGYPEAWFSRDFEATGPFFEREVYEYPNHQQACTLWYHDHAMALTRLNVYAGLA
GFYLISDAFEKSLELPKGEYDIPLMIMDRTFQEDGALFYPSRPNNTPEDSDIPDPSIVPFFCGETILVNGKVWPYLEVEPRKYRFRILNASNTRTYELHL
DNDATILQIGSDGGFLPRPVHHQSFSIAPAERFDVIIDFSAYENKTITLKNKAGCGQEVNPETDANIMQFKVTRPLKGRAPKTLRPIFKPLPPLRPCRAD
KERTLTLTGTQDKYGRPILLLDNQFWNDPVTENPRLGSVEVWSIVNPTRGTHPIHLHLVQFRVIDRRPFDTEVYQSTGDIVYTGPNEAPPLHEQGYKDTI
QAHAGEVIRIIARFVPYSGRYVWHCHILEHEDYDMMRPMDIITGSAWSHPQFEKGENLYFQSGTGPGQPPFPPPPPFTPPPPQTPNGASGENSTGAIAGG
VAAGAALLFAAPAIGFAWWRRRRPIEATGGGSGGGSGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVT
TLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLSPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYKTG*
Sequence features: (with their position in the amino acid sequence, see the list of supported features)
RFC25 scar (shown in bold): 33 to 34, 161 to 162, 543 to 544, 564 to 565, 628 to 629, 638 to 639
Strep-tag II: 547 to 554
TEV cleavage site: 556 to 562
Amino acid composition:
Ala (A)51 (5.8%)
Arg (R)40 (4.6%)
Asn (N)41 (4.7%)
Asp (D)50 (5.7%)
Cys (C)7 (0.8%)
Gln (Q)26 (3.0%)
Glu (E)59 (6.7%)
Gly (G)75 (8.5%)
His (H)34 (3.9%)
Ile (I)48 (5.5%)
Leu (L)68 (7.7%)
Lys (K)48 (5.5%)
Met (M)16 (1.8%)
Phe (F)43 (4.9%)
Pro (P)76 (8.7%)
Ser (S)37 (4.2%)
Thr (T)58 (6.6%)
Trp (W)14 (1.6%)
Tyr (Y)34 (3.9%)
Val (V)53 (6.0%)
Amino acid counting
Total number:878
Positively charged (Arg+Lys):88 (10.0%)
Negatively charged (Asp+Glu):109 (12.4%)
Aromatic (Phe+His+Try+Tyr):125 (14.2%)
Biochemical parameters
Atomic composition:C4456H6781N1195O1293S23
Molecular mass [Da]:98518.5
Theoretical pI:5.91
Extinction coefficient at 280 nm [M-1 cm-1]:127660 / 128098 (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 (0.72)good (0.75)good (0.71)good (0.79)good (0.79)good (0.69)
Alignments (obtained from PredictProtein.org)
SwissProt:P42212 (98% identity on 238 AAs), P07788 (67% identity on 508 AAs)
TrEML:B4AIB1 (99% identity on 509 AAs), A8FAG9 (98% identity on 507 AAs), Q71RY9 (98% identity on 238 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:cytosol (100%)
Bacteria:secreted (63%)
Eukarya:mitochondria (21%)
Gene Ontology (reliability in brackets)
Molecular Function Ontology:GO:0005507 (37%), GO:0005515 (23%)
Biological Process Ontology:GO:0055114 (30%), GO:0006091 (27%)
 
Predicted features:
Disulfid bridges: -
Transmembrane helices:601 to 618 going inwards
The BioBrick-AutoAnnotator was created by TU-Munich 2013 iGEM team. For more information please see the documentation.
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Categories
Parameters
None