BBa_K1982010

BBa_K1982010 Version 1

Component

Source:
http://parts.igem.org/Part:BBa_K1982010
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Zexu Li
Date created: 2016-10-08 11:00:00
Date modified: 2016-10-18 10:53:31

CRY2-VP64(Eukaryotic LACE system)



    • Details

    Types
    DnaRegion

    Roles
    CDS

    Coding

    Sequences BBa_K1982010_sequence (Version 1)

    Description

    {| style="color:black" cellpadding="6" cellspacing="1" border="2" align="right"
    ! colspan="2" style="background:#FFBF00;"|RBS-CRY2-VP64-HA-FLAG
    |-
    |Function
    |gene activation
    |-
    |Use in
    |Prokaryotic cells
    |-
    |RFC standard
    |RFC 10
    |-
    |Backbone
    |pSB1C3

    |-
    |Submitted by
    |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 VP64 fused to its C 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.

    We used the full-length CRY2 (BBa_K1982002) and fused it to the transcription activator domain VP64 (BBa_K1982000). An prokaryotic RBS sequence from the Community collection(BBa_B0034) fused to the beginning of CRY2-VP64. For detection of expression the fusion protein was tagged with a FLAG-epitope coding sequence (gactacaaggacgacgacgacaaa) .Figure 1 illustrates the detailed design of the whole 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)
     ATGAAGATG ... GACGACAAATAATAA
     ORF from nucleotide position 1 to 2076 (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     		MKMDKKTIVWFRRDLRIEDNPALAAAAHEGSVFPVFIWCPEEEGQFYPGRASRWWMKQSLAHLSQSLKALGSDLTLIKTHNTISAILDCIRVTGATKVVF
    NHLYDPVSLVRDHTVKEKLVERGISVQSYNGDLLYEPWEIYCEKGKPFTSFNSYWKKCLDMSIESVMLPPPWRLMPITAAAEAIWACSIEELGLENEAEK
    PSNALLTRAWSPGWSNADKLLNEFIEKQLIDYAKNSKKVVGNCTSLLSPYLHFGEISVRHVFQCARMKQIIWARDKNSEGEESADLFLRGIGLREYSRYI
    CFNFPFTHEQSLLSHLRFFPWDADVDKFKAWRQGRTGYPLVDAGMRELWATGWMHNRIRVIVSSFAVKFLLLPWKWGMKYFWDTLLDADLECDILGWQYI
    SGSIPDGHELDRLDNPALQGAKYDPEGEYIRQWLPELARLPTEWIHHPWDAPLTVLKASGVELGTNYAKPIVDIDTARELLAKAISRTREAQIMIGAAPD
    EIVADSFEALGANTIKEPGLCPSVSSNDQQVPSAVRYNGSKRVKPEEEEERDMKKSRGFDERELFSTAESSSSSSVFFVSQSCSLASEGKNLEGIQDSSD
    QITTSLGKNGCKPKKKRKVGRADALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLINYPYDVPDYASDYKDDDDK*
    Sequence features: (with their position in the amino acid sequence, see the list of supported features)
    SV40 nuclear localization sequence: 613 to 619
    HA-tag: 675 to 683
    Flag-tag: 685 to 692
    Enterokinase cleavage site: 688 to 692
    Amino acid composition:
    Ala (A)53 (7.7%)
    Arg (R)35 (5.1%)
    Asn (N)22 (3.2%)
    Asp (D)62 (9.0%)
    Cys (C)12 (1.7%)
    Gln (Q)18 (2.6%)
    Glu (E)48 (6.9%)
    Gly (G)40 (5.8%)
    His (H)13 (1.9%)
    Ile (I)37 (5.3%)
    Leu (L)71 (10.3%)
    Lys (K)44 (6.4%)
    Met (M)16 (2.3%)
    Phe (F)29 (4.2%)
    Pro (P)34 (4.9%)
    Ser (S)58 (8.4%)
    Thr (T)24 (3.5%)
    Trp (W)22 (3.2%)
    Tyr (Y)21 (3.0%)
    Val (V)33 (4.8%)
    Amino acid counting
    Total number:692
    Positively charged (Arg+Lys):79 (11.4%)
    Negatively charged (Asp+Glu):110 (15.9%)
    Aromatic (Phe+His+Try+Tyr):85 (12.3%)
    		Biochemical parameters
    Atomic composition:C3518H5403N929O1056S28
    Molecular mass [Da]:78505.9
    Theoretical pI:4.93
    Extinction coefficient at 280 nm [M-1 cm-1]:152290 / 153040 (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 (0.69)good (0.73)good (0.68)good (0.76)excellent (0.80)good (0.68)
    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.



    Notes

    no

    Source

    CRY2 and CIB1 are extracted from Arabidopsis thaliana. tCas9 is from GE Share company. VP64 is synthetic construct.

    Sequence Annotation Location Component / Role(s)
    Eukaryotic rbs
    ATG
    CRY2
    SV40 NLS
    VP16
    VP16
    VP16
    VP16
    Linker
    HA
    FLAG
    TAA
    		1,18
    19,21
    22,1854
    1855,1875
    1876,1914
    1915,1953
    1954,1992
    1993,2031
    2032,2037
    2037,2063
    2071,2094
    2095,2100
    		ribosome_entry_site feature/rbs
    feature/start start_codon
    feature/protein CDS
    feature/misc sequence_feature
    sequence_feature feature/misc
    sequence_feature feature/misc
    sequence_feature feature/misc
    feature/misc sequence_feature
    sequence_feature feature/misc
    feature/tag tag
    feature/tag tag
    feature/stop stop_codon

    igem#status
    Planning
     
    synbiohub#ownedBy
    user/james
     
    synbiohub#ownedBy
    user/myers
     
    synbiohub#topLevel
    BBa_K1982010/1
     

    Attachments

    © 2018 Newcastle University, University of Utah, and collaborators
    About SynBioHub | View Source on Github | Report an Issue
    | v1.6.1 (86615526)
    ","mutableDescription":"{| style=\"color:black\" cellpadding=\"6\" cellspacing=\"1\" border=\"2\" align=\"right\"
    \n! colspan=\"2\" style=\"background:#FFBF00;\"|RBS-CRY2-VP64-HA-FLAG
    \n|-
    \n|Function
    \n|gene activation
    \n|-
    \n|Use in
    \n|Prokaryotic cells
    \n|-
    \n|RFC standard
    \n|RFC 10
    \n|-
    \n|Backbone
    \n|pSB1C3

    \n|-
    \n|Submitted by
    \n|NEU-China 2016
    \n|}
    \n
    \nThe 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 VP64 fused to its C 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.
    \n
    \nWe used the full-length CRY2 (BBa_K1982002) and fused it to the transcription activator domain VP64 (BBa_K1982000). An prokaryotic RBS sequence from the Community collection(BBa_B0034) fused to the beginning of CRY2-VP64. For detection of expression the fusion protein was tagged with a FLAG-epitope coding sequence (gactacaaggacgacgacgacaaa) .Figure 1 illustrates the detailed design of the whole device.
    \n
    \n
    \n
    \n
    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)
     ATGAAGATG ... GACGACAAATAATAA
     ORF from nucleotide position 1 to 2076 (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     		MKMDKKTIVWFRRDLRIEDNPALAAAAHEGSVFPVFIWCPEEEGQFYPGRASRWWMKQSLAHLSQSLKALGSDLTLIKTHNTISAILDCIRVTGATKVVF
    NHLYDPVSLVRDHTVKEKLVERGISVQSYNGDLLYEPWEIYCEKGKPFTSFNSYWKKCLDMSIESVMLPPPWRLMPITAAAEAIWACSIEELGLENEAEK
    PSNALLTRAWSPGWSNADKLLNEFIEKQLIDYAKNSKKVVGNCTSLLSPYLHFGEISVRHVFQCARMKQIIWARDKNSEGEESADLFLRGIGLREYSRYI
    CFNFPFTHEQSLLSHLRFFPWDADVDKFKAWRQGRTGYPLVDAGMRELWATGWMHNRIRVIVSSFAVKFLLLPWKWGMKYFWDTLLDADLECDILGWQYI
    SGSIPDGHELDRLDNPALQGAKYDPEGEYIRQWLPELARLPTEWIHHPWDAPLTVLKASGVELGTNYAKPIVDIDTARELLAKAISRTREAQIMIGAAPD
    EIVADSFEALGANTIKEPGLCPSVSSNDQQVPSAVRYNGSKRVKPEEEEERDMKKSRGFDERELFSTAESSSSSSVFFVSQSCSLASEGKNLEGIQDSSD
    QITTSLGKNGCKPKKKRKVGRADALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLINYPYDVPDYASDYKDDDDK*
    Sequence features: (with their position in the amino acid sequence, see the list of supported features)
    SV40 nuclear localization sequence: 613 to 619
    HA-tag: 675 to 683
    Flag-tag: 685 to 692
    Enterokinase cleavage site: 688 to 692
    Amino acid composition:
    Ala (A)53 (7.7%)
    Arg (R)35 (5.1%)
    Asn (N)22 (3.2%)
    Asp (D)62 (9.0%)
    Cys (C)12 (1.7%)
    Gln (Q)18 (2.6%)
    Glu (E)48 (6.9%)
    Gly (G)40 (5.8%)
    His (H)13 (1.9%)
    Ile (I)37 (5.3%)
    Leu (L)71 (10.3%)
    Lys (K)44 (6.4%)
    Met (M)16 (2.3%)
    Phe (F)29 (4.2%)
    Pro (P)34 (4.9%)
    Ser (S)58 (8.4%)
    Thr (T)24 (3.5%)
    Trp (W)22 (3.2%)
    Tyr (Y)21 (3.0%)
    Val (V)33 (4.8%)
    Amino acid counting
    Total number:692
    Positively charged (Arg+Lys):79 (11.4%)
    Negatively charged (Asp+Glu):110 (15.9%)
    Aromatic (Phe+His+Try+Tyr):85 (12.3%)
    		Biochemical parameters
    Atomic composition:C3518H5403N929O1056S28
    Molecular mass [Da]:78505.9
    Theoretical pI:4.93
    Extinction coefficient at 280 nm [M-1 cm-1]:152290 / 153040 (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 (0.69)good (0.73)good (0.68)good (0.76)excellent (0.80)good (0.68)
    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.


    \n","mutableNotesSource":"no","mutableNotes":"no
    \n","sourceSource":"CRY2 and CIB1 are extracted from Arabidopsis thaliana. tCas9 is from GE Share company. VP64 is synthetic construct.","source":"CRY2 and CIB1 are extracted from Arabidopsis thaliana. tCas9 is from GE Share company. VP64 is synthetic construct.
    \n","description":"CRY2-VP64(Eukaryotic LACE system)
    \n","comments":[],"labels":[],"canEdit":false,"canUpdate":false,"annotations":[{"type":"uri","name":"igem#status","value":"Planning","nameDef":"http://wiki.synbiohub.org/wiki/Terms/igem#status","id":"igem#status","uri":"http://wiki.synbiohub.org/wiki/Terms/igem#status/Planning","url":"http://wiki.synbiohub.org/wiki/Terms/igem#status/Planning"},{"type":"uri","name":"synbiohub#ownedBy","value":"user/james","nameDef":"http://wiki.synbiohub.org/wiki/Terms/synbiohub#ownedBy","id":"synbiohub#ownedBy","uri":"https://synbiohub.org/user/james","url":"/user/james"},{"type":"uri","name":"synbiohub#ownedBy","value":"user/myers","nameDef":"http://wiki.synbiohub.org/wiki/Terms/synbiohub#ownedBy","id":"synbiohub#ownedBy","uri":"https://synbiohub.org/user/myers","url":"/user/myers"},{"type":"uri","name":"synbiohub#topLevel","value":"BBa_K1982010/1","nameDef":"http://wiki.synbiohub.org/wiki/Terms/synbiohub#topLevel","id":"synbiohub#topLevel","uri":"https://synbiohub.org/public/igem/BBa_K1982010/1","url":"/public/igem/BBa_K1982010/1"}],"triplestore":"public","graphUri":null,"remote":null,"large":false,"encodedProteins":[],"typeFilter":"Show Only Root Objects"}