BBa_K1982011

BBa_K1982011 Version 1

Component

Source:
http://parts.igem.org/Part:BBa_K1982011
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 10:57:53

tCas9-Vp64(Eukaryoticc)



    • Details

    Types
    DnaRegion

    Roles
    Coding

    CDS

    Sequences BBa_K1982011_sequence (Version 1)

    Description

    {| style="color:black" cellpadding="6" cellspacing="1" border="2" align="right"
    ! colspan="2" style="background:#FFBF00;"|RBS-tCas9-VP64-HA-FLAG
    |-
    |Function
    |gene activation
    |-
    |Use in
    |Eukaryotic 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 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.

    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).





    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.



    Notes

    mutant Pst 1 :gctacctgcaG to gctacctgcaC
    mutant Pst 1 :actatctgcaG to actatctgcaC

    Source

    tCas9 is from GE Share company. VP64 is synthetic construct.

    Sequence Annotation Location Component / Role(s)
    Eukaryotic rbs
    ATG
    tCas9
    SV40 NLS
    VP16
    VP16
    VP16
    VP16
    Linker
    HA
    FLAG
    TAATAA
    		1,18
    19,21
    22,4122
    4123,4143
    4144,4182
    4183,4221
    4222,4260
    4261,4299
    4300,4305
    4306,4332
    4339,4362
    4363,4368
    		ribosome_entry_site feature/rbs
    start_codon feature/start
    CDS feature/protein
    sequence_feature feature/misc
    feature/misc sequence_feature
    feature/misc sequence_feature
    feature/misc sequence_feature
    feature/misc sequence_feature
    feature/misc sequence_feature
    feature/tag tag
    tag feature/tag
    stop_codon feature/stop

    igem#status
    Planning
     
    synbiohub#ownedBy
    user/james
     
    synbiohub#ownedBy
    user/myers
     
    synbiohub#topLevel
    BBa_K1982011/1
     

    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-tCas9-VP64-HA-FLAG
    \n|-
    \n|Function
    \n|gene activation
    \n|-
    \n|Use in
    \n|Eukaryotic 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 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.
    \n
    \ntCas9 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).
    \n
    \n
    \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)
     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.


    \n","mutableNotesSource":"mutant Pst 1 :gctacctgcaG to gctacctgcaC\nmutant Pst 1 :actatctgcaG to actatctgcaC","mutableNotes":"mutant Pst 1 :gctacctgcaG to gctacctgcaC
    \nmutant Pst 1 :actatctgcaG to actatctgcaC
    \n","sourceSource":"tCas9 is from GE Share company. VP64 is synthetic construct.","source":"tCas9 is from GE Share company. VP64 is synthetic construct.
    \n","description":"tCas9-Vp64(Eukaryoticc)
    \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_K1982011/1","nameDef":"http://wiki.synbiohub.org/wiki/Terms/synbiohub#topLevel","id":"synbiohub#topLevel","uri":"https://synbiohub.org/public/igem/BBa_K1982011/1","url":"/public/igem/BBa_K1982011/1"}],"triplestore":"public","graphUri":null,"remote":null,"large":false,"encodedProteins":[],"typeFilter":"Show Only Root Objects"}