Device

Part:BBa_K1982006

Designed by: Zexu Li   Group: iGEM16_NEU-China   (2016-09-12)
Revision as of 13:41, 15 October 2016 by King (Talk | contribs)


tCas9-Vp64(Prokaryotic)

RBS-tCas9-VP64-HA-FLAG
Function gene activation
Use in Prokaryotic cells
RFC standard RFC 10
Backbone pSB1C3
Submitted by [http://2016.igem.org/Team:NEU-China NEU-China 2016]

The CRY2/CIBN interaction is entirely genetically encoded. The binding reverses within minutes in the dark, allowing rapid shutoff of transcription by placing samples in the dark. This fusion protein is for use in LACE(light-activated CRISPR/Cas9 effector) system, and a tCas9 fused to its N terminus. To regulate DNA transcription by blue light, the system is based on CRY2/CIBN interaction in which a light-mediated protein interaction brings together two protein (tCas9 and an activation domain VP64) . If we remove the stimulation of blue light, dark reversion of CRY2 will dissociate the interaction with CIBN and shut off transcription.

Figure 1: Construct design.
tCas9 can be tagged with transcriptional activators, and targeting these dCas9 fusion proteins to the promoter region results in robust transcription activation of downstream target genes. This tCas9-based activators is the case that tCas9 fused directly to a single transcriptional activator( VP64).
Figure 2: Figure 1 illustrates the detailed design of LACE device



Protein data table for BioBrick BBa_ automatically created by the BioBrick-AutoAnnotator version 1.0
Nucleotide sequence in RFC 10: (underlined part encodes the protein)
 ATGGACAAG ... GACGACAAATAATAA
 ORF from nucleotide position 1 to 4344 (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 
901 
1001 
1101 
1201 
1301 
1401 		MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLHEIFSNEMAKVDDSFFHR
LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP
INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI
LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK
NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI
IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD
SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP
VENTQLQNEKLYLYYLHNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL
TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK
YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV
QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK
YSLFELENGRKRMLARAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDK
PIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDPKKKRKVGRADALDDFDLDMLGSDALDDFDLD
MLGSDALDDFDLDMLGSDALDDFDLDMLINYPYDVPDYASDYKDDDDK*
Sequence features: (with their position in the amino acid sequence, see the list of supported features)
RFC25 scar (shown in bold): 657 to 658, 1102 to 1103
SV40 nuclear localization sequence: 1369 to 1375
HA-tag: 1431 to 1439
Flag-tag: 1441 to 1448
Enterokinase cleavage site: 1444 to 1448
Amino acid composition:
Ala (A)80 (5.5%)
Arg (R)80 (5.5%)
Asn (N)71 (4.9%)
Asp (D)125 (8.6%)
Cys (C)2 (0.1%)
Gln (Q)50 (3.5%)
Glu (E)108 (7.5%)
Gly (G)73 (5.0%)
His (H)33 (2.3%)
Ile (I)94 (6.5%)
Leu (L)160 (11.0%)
Lys (K)156 (10.8%)
Met (M)26 (1.8%)
Phe (F)67 (4.6%)
Pro (P)38 (2.6%)
Ser (S)79 (5.5%)
Thr (T)65 (4.5%)
Trp (W)7 (0.5%)
Tyr (Y)59 (4.1%)
Val (V)75 (5.2%)
Amino acid counting
Total number:1448
Positively charged (Arg+Lys):236 (16.3%)
Negatively charged (Asp+Glu):233 (16.1%)
Aromatic (Phe+His+Try+Tyr):166 (11.5%)
Biochemical parameters
Atomic composition:C7507H11924N2038O2239S28
Molecular mass [Da]:167451.2
Theoretical pI:8.02
Extinction coefficient at 280 nm [M-1 cm-1]:126410 / 126535 (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 (0.66)good (0.70)acceptable (0.60)good (0.71)excellent (0.84)excellent (0.85)
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.

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
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 2758
    Illegal NgoMIV site found at 3667
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 3786
    Illegal SapI.rc site found at 1177
    Illegal SapI.rc site found at 1419


[edit]
Categories
//function/crispr
//function/crispr/cas9
Parameters
directionForward