Coding

Part:BBa_K1150002

Designed by: Freiburg 2013   Group: iGEM13_Freiburg   (2013-09-16)

KRAB

KRAB
Function Repressor domain
Use in Mammalian cells
Organism Homo sapiens
RFC standard RFC 25
Backbone pSB1C3
Source Konrad Müller
Submitted by [http://2013.igem.org/Team:Freiburg Freiburg 2013]
Fig. 1 Structural Mechanism of KRAB, as suggested Urrutia, R., 2003.

Krüppel-associated Box repressor domains - commonly termed as KRAB - are highly conserved polypeptide motifs and were first functionally characterized in 1991 (Rosati et al., 1991). As they constitute about one third of all human zinc finger transcription factors, key regulatory features in higher eukaryotic transcrip-
tomics are suggested (Witzgall et al., 1994). Even in terms of tetrapod evolution, the role of their great abundance has been extensively discussed (Birtle, 2006). Even though KRAB minimal domains are usually no longer than 50-75 amino acids, their mechanism of function remains complex.


Common biochemical models suggest a key role in epigenetic silencing, by recruiting a scaffold of diverse proteins - amongst others histone deacetylases and histone methyltransferases (Urrutia, 2003). Til date in 2013, KRAB repressor domains were attached to several DNA binding proteins such as tetR, TAL effec-
tors and [http://2013.igem.org/Team:Freiburg/Project/effector#repression dCas9] - thereby efficiently silencing gene expression downstream of desired target promoters.

The latter combinatorial attempt was conducted twice by Team Freiburg 2013, which resulted in two composite parts - SV40-dCas9-KRAB and CMV-dCas9-KRAB.

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.rc site found at 244
    Illegal SapI.rc site found at 208


Protein data table for BioBrick BBa_K1150002 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)
 ATGGATGCT ... GATCTCTGGACCGGT
 ORF from nucleotide position 1 to 372 (excluding stop-codon)
Amino acid sequence: (RFC 25 scars in shown in bold, other sequence features underlined; both given below)
1 
101 		MDAKSLTAWSRTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGYQLTKPDVILRLEKGEEPWLVEREIHQETHPDSETAFEIKSSVSSR
SIFKDKQSCDIKMEGMARNDLWTG*
Sequence features: (with their position in the amino acid sequence, see the list of supported features)
None of the supported features appeared in the sequence
Amino acid composition:
Ala (A)5 (4.0%)
Arg (R)7 (5.6%)
Asn (N)4 (3.2%)
Asp (D)9 (7.3%)
Cys (C)1 (0.8%)
Gln (Q)5 (4.0%)
Glu (E)12 (9.7%)
Gly (G)4 (3.2%)
His (H)2 (1.6%)
Ile (I)6 (4.8%)
Leu (L)12 (9.7%)
Lys (K)10 (8.1%)
Met (M)4 (3.2%)
Phe (F)5 (4.0%)
Pro (P)3 (2.4%)
Ser (S)10 (8.1%)
Thr (T)9 (7.3%)
Trp (W)4 (3.2%)
Tyr (Y)3 (2.4%)
Val (V)9 (7.3%)
Amino acid counting
Total number:124
Positively charged (Arg+Lys):17 (13.7%)
Negatively charged (Asp+Glu):21 (16.9%)
Aromatic (Phe+His+Try+Tyr):14 (11.3%)
Biochemical parameters
Atomic composition:C647H1016N172O198S5
Molecular mass [Da]:14532.5
Theoretical pI:5.22
Extinction coefficient at 280 nm [M-1 cm-1]:26470 / 26533 (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 (0.70)good (0.65)good (0.76)excellent (0.85)good (0.79)
Alignments (obtained from PredictProtein.org)
   There were no alignments for this protein in the data base. The BLAST search was initialized and should be ready in a few hours.
Predictions (obtained from PredictProtein.org)
   There were no predictions for this protein in the data base. The prediction was initialized and should be ready in a few hours.
The BioBrick-AutoAnnotator was created by TU-Munich 2013 iGEM team. For more information please see the documentation.
If you have any questions, comments or suggestions, please leave us a comment.

References

Rosati, M. et al. (1991). Members of the zinc finger protein gene family sharing a conserved N-terminal module. Nucleic acids research 19, 5661-5667.
Witzgall, R. et al. (1994). The Krüppel-associated box-A domain of zinc finger proteins mediates transcriptional repression. Proc Nati Acad Sci 91, 4514-4518.
Birtle, Z. and Ponting, C. (2006). Meisetz and the birth of the KRAB motif. Bioinformatics 22, 2841-2845.
Urrutia, R. (2003). KRAB-containing zinc-finger repressor proteins. Genome Biology 4, 4:231.

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Categories
Parameters
None