//function/crispr
type: Collection
persistent identity: function crispr
display id: function_crispr
version: 1
was generated by: https://synbiohub.org/public/igem/igem2sbol/1
owned by: james
owned by: myers
member: BBa_K1026001
member: BBa_K2017007
member: Prokaryoti
member: BBa_K1150050
member: BBa_K1160000
member: BBa_K1559005
member: BBa_K1137014
member: BBa_K2017010
member: BBa_K1150017
member: BBa_K1150000
member: BBa_K2017008
member: BBa_K1982007
member: TC
member: BBa_K2017014
member: BBa_K1137013
member: BBa_K1129006
member: BBa_K1179002
member: BBa_K2017001
member: BBa_K1026000
member: Cse1
member: BBa_K1982004
member: BBa_K2017009
member: BBa_K1559004
member: BBa_K1982008
member: BBa_K1559008
member: VP64
member: BBa_K2017013
member: BBa_K2017000
member: BBa_K1994013
member: BBa_K1982003
member: BBa_K1994019
member: BBa_K2017012
member: BBa_K1559003
member: BBa_K1982009
member: BBa_K1081000
member: BBa_K1559002
member: BBa_K1982005
member: BBa_K1559007
member: BBa_K1982011
member: Const gR4m
member: //function/crispr/cas9
member: BBa_K1218011
member: BBa_K1982002
member: BBa_K2017011
member: BBa_K1559006
member: BBa_K1982010
member: BBa_K1982006

//function/crispr/cas9
type: Collection
persistent identity: function crispr cas9
display id: function_crispr_cas9
version: 1
was generated by: https://synbiohub.org/public/igem/igem2sbol/1
owned by: james
owned by: myers
member: BBa_K1026001
member: https://synbiohub.org/public/igem/BBa_K1994016/1
member: Prokaryoti
member: BBa_K1150050
member: BBa_K1160000
member: BBa_K1559005
member: BBa_K1137014
member: BBa_K1150017
member: https://synbiohub.org/public/igem/BBa_K1994017/1
member: BBa_K1150000
member: BBa_K1982007
member: https://synbiohub.org/public/igem/BBa_K1994015/1
member: https://synbiohub.org/public/igem/BBa_K1946002/1
member: TC
member: BBa_K1129006
member: BBa_K1179002
member: BBa_K1026000
member: BBa_K1982004
member: BBa_K1559004
member: BBa_K1982008
member: https://synbiohub.org/public/igem/BBa_K1994044/1
member: BBa_K1559008
member: VP64
member: https://synbiohub.org/public/igem/BBa_K1774001/1
member: BBa_K1994013
member: BBa_K1559003
member: BBa_K1081000
member: BBa_K1559002
member: BBa_K1559007
member: BBa_K1982011
member: https://synbiohub.org/public/igem/BBa_K1994021/1
member: BBa_K1218011
member: https://synbiohub.org/public/igem/BBa_K1994025/1
member: BBa_K1559006
member: BBa_K1982006

BBa_K1026000
type: ComponentDefinition
persistent identity: BBa K1026000
display id: BBa_K1026000
version: 1
was generated by: https://synbiohub.org/public/igem/igem2sbol/1
description: Constitutively Expressed dCas9 Operon
creator: Hongyi WU
created: 2013-09-15T11:00:00Z
modified: 2015-05-08T01:08:46Z
discontinued: false
dominant: false
experience: experience/Works
group u list: _1333_
m user id: 0
owner id: 12098
owning group id: 9
sample status: It's complicated
status: status/Planning
bookmark: true
mutable description: This Constitutively Expressed dCas9 Operon is an assembly of the constitutive promoter BBa_J23100, a strong RBS BBa_B0034, coding sequence of dCas9 protein and a reliable terminator BBa_B0015. dCas9 is a mutant of Cas9, with its endonuclease activity wiped out. dCas9 acts as the sole protein in CRISPRi system (Qi et al., 2013). Reference QI, LEI S., LARSON, MATTHEW H., GILBERT, LUKE A., DOUDNA, JENNIFER A., WEISSMAN, JONATHAN S., ARKIN, ADAM P. & LIM, WENDELL A. 2013. Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression. Cell, 152, 1173-1183.
mutable notes: The expression level of dCas9 shall be delicately controlled, so as to acquire continuous adjustment (regulation) of target genes.
mutable provenance: The dCas9 comes from S. pyogenes.
owned by: james
owned by: myers
star: false
type: DnaRegion
role: SO:0000804
role: partType/Composite
component: BBa_B0034
component: BBa_B0015
component: BBa_K1026001
component: BBa_J23100
sequence annotation: BBa_J23100
sequence annotation: BBa_B0015
sequence annotation: BBa_B0034
sequence annotation: BBa_K1026001
sequence: https://synbiohub.org/public/igem/BBa_K1026000_sequence/1

BBa_B0034
type: Component
persistent identity: component2347321
display id: component2347321
version: 1
top level: BBa_K1026000
access: public
definition: https://synbiohub.org/public/igem/BBa_B0034/1

BBa_B0015
type: Component
persistent identity: component2347329
display id: component2347329
version: 1
top level: BBa_K1026000
access: public
definition: https://synbiohub.org/public/igem/BBa_B0015/1

BBa_K1026001
type: Component
persistent identity: component2347322
display id: component2347322
version: 1
top level: BBa_K1026000
access: public
definition: BBa_K1026001

BBa_J23100
type: Component
persistent identity: component2347319
display id: component2347319
version: 1
top level: BBa_K1026000
access: public
definition: https://synbiohub.org/public/igem/BBa_J23100/1

BBa_J23100
type: SequenceAnnotation
persistent identity: annotation2347319
display id: annotation2347319
version: 1
top level: BBa_K1026000
location: https://synbiohub.org/public/igem/BBa_K1026000/annotation2347319/range2347319/1
component: BBa_J23100

https://synbiohub.org/public/igem/BBa_K1026000/annotation2347319/range2347319/1
type: Range
persistent identity: range2347319
display id: range2347319
version: 1
direction: direction/reverse
top level: BBa_K1026000
start: 1
end: 35
orientation: inline

BBa_B0015
type: SequenceAnnotation
persistent identity: annotation2347329
display id: annotation2347329
version: 1
top level: BBa_K1026000
location: https://synbiohub.org/public/igem/BBa_K1026000/annotation2347329/range2347329/1
component: BBa_B0015

https://synbiohub.org/public/igem/BBa_K1026000/annotation2347329/range2347329/1
type: Range
persistent identity: range2347329
display id: range2347329
version: 1
direction: direction/reverse
top level: BBa_K1026000
start: 4183
end: 4311
orientation: inline

BBa_B0034
type: SequenceAnnotation
persistent identity: annotation2347321
display id: annotation2347321
version: 1
top level: BBa_K1026000
location: https://synbiohub.org/public/igem/BBa_K1026000/annotation2347321/range2347321/1
component: BBa_B0034

https://synbiohub.org/public/igem/BBa_K1026000/annotation2347321/range2347321/1
type: Range
persistent identity: range2347321
display id: range2347321
version: 1
direction: direction/reverse
top level: BBa_K1026000
start: 44
end: 55
orientation: inline

BBa_K1026001
type: SequenceAnnotation
persistent identity: annotation2347322
display id: annotation2347322
version: 1
top level: BBa_K1026000
location: https://synbiohub.org/public/igem/BBa_K1026000/annotation2347322/range2347322/1
component: BBa_K1026001

https://synbiohub.org/public/igem/BBa_K1026000/annotation2347322/range2347322/1
type: Range
persistent identity: range2347322
display id: range2347322
version: 1
direction: direction/reverse
top level: BBa_K1026000
start: 62
end: 4174
orientation: inline

Cse1
type: ComponentDefinition
persistent identity: BBa K1218004
display id: BBa_K1218004
version: 1
was generated by: https://synbiohub.org/public/igem/igem2sbol/1
description: CRISPR CasA (Ancestral)
creator: Trevor Kalkus, Gordon Wade, Alissa Greenberg
created: 2013-08-27T11:00:00Z
modified: 2015-05-08T01:09:42Z
discontinued: false
dominant: false
experience: experience/None
group u list: _1532_
m user id: 0
owner id: 17712
owning group id: 9
sample status: It's complicated
status: status/Planning
bookmark: false
mutable description: The E. coli CRISPR system uses the CASCADE complex to target invasive DNA. CasA is the first subunit of the CASCADE complex and is responsible for the initial identification of invasive DNA. The ancestral version of this has been reconstructed using the seed sequences from the PFAM data base by creating a phylogenetic tree using PhyML and then using the WAG method in PAML with Lazarus. This part can be used with casBCDE to create the full CASCADE protein complex.
mutable notes: None
mutable provenance: DNA2.0
owned by: james
owned by: myers
star: false
type: DnaRegion
role: partType/Coding
role: SO:0000316
sequence: https://synbiohub.org/public/igem/BBa_K1218004_sequence/1

BBa_K1026001
type: ComponentDefinition
persistent identity: BBa K1026001
display id: BBa_K1026001
version: 1
was generated by: https://synbiohub.org/public/igem/igem2sbol/1
description: dCas9
creator: Hongyi WU
created: 2013-09-15T11:00:00Z
modified: 2015-05-08T01:08:46Z
discontinued: false
dominant: false
experience: experience/Works
group u list: _1333_
m user id: 0
owner id: 12098
owning group id: 9
sample status: Not in stock
status: status/Unavailable
bookmark: false
mutable description: dCas9 is a mutant of Cas9, with its endonuclease activity wiped out. dCas9 acts as the sole protein in CRISPRi system (Qi et al., 2013). Reference QI, LEI S., LARSON, MATTHEW H., GILBERT, LUKE A., DOUDNA, JENNIFER A., WEISSMAN, JONATHAN S., ARKIN, ADAM P. & LIM, WENDELL A. 2013. Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression. Cell, 152, 1173-1183.
mutable notes: Mutation: Aspartate 10 to Alanine (D10A) and Histidine 840 to Alanine (H840A) in order to cripple the endonuclease activity.
mutable provenance: The dCas9 comes from S. pyogenes.
owned by: james
owned by: myers
star: false
type: DnaRegion
role: partType/Coding
role: SO:0000316
sequence: https://synbiohub.org/public/igem/BBa_K1026001_sequence/1

BBa_K1982004
type: ComponentDefinition
persistent identity: BBa K1982004
display id: BBa_K1982004
version: 1
was generated by: https://synbiohub.org/public/igem/igem2sbol/1
description: tCas9-CIBN (Prokaryotic LACE system)
creator: Zexu Li
created: 2016-09-11T11:00:00Z
modified: 2016-10-18T10:12:30Z
discontinued: false
dominant: false
experience: experience/Works
group u list: _2449_
m user id: 30176
owner id: 30176
owning group id: 9
status: status/Planning
bookmark: false
mutable description: Optogenetic systems enable precise spatial and temporal control of cell behavior. A light-activated CRISPR/Cas9 effector (LACE) system that induces transcription of endogenous genes in the presence of blue light.This was accomplished by fusing the light-inducible heterodimerizing proteins CRY2 and CIB1 to a transactivation domain and the catalytically inactive tCas9, respectively. The versatile LACE system can be easily directed to new DNA sequences for the dynamic regulation of endogenous genes[1]. we fused the light-inducible heterodimerizering proteins CRY2 and CIB1 from Arabidopsis thaliana to the VP64 transactivation domain and C-terminus of tCas9.
mutable notes: 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.
mutable provenance: CRY2 and CIB1 are extracted from Arabidopsis thaliana. tCas9 is from GE Share company. VP64 is synthetic construct.
owned by: james
owned by: myers
star: false
type: DnaRegion
role: partType/Device
role: SO:0000804
sequence annotation: ATG
sequence annotation: CIBN
sequence annotation: HA
sequence annotation: Prokaryotic rbs
sequence annotation: tCas9
sequence annotation: TAATAA
sequence: https://synbiohub.org/public/igem/BBa_K1982004_sequence/1

ATG
type: SequenceAnnotation
persistent identity: annotation2483005
display id: annotation2483005
version: 1
top level: BBa_K1982004
role: SO:0000318
role: feature/start
location: https://synbiohub.org/public/igem/BBa_K1982004/annotation2483005/range2483005/1

https://synbiohub.org/public/igem/BBa_K1982004/annotation2483005/range2483005/1
type: Range
persistent identity: range2483005
display id: range2483005
version: 1
direction: direction/forward
top level: BBa_K1982004
start: 4105
end: 4107
orientation: inline

CIBN
type: SequenceAnnotation
persistent identity: annotation2483007
display id: annotation2483007
version: 1
top level: BBa_K1982004
role: SO:0000316
role: feature/protein
location: https://synbiohub.org/public/igem/BBa_K1982004/annotation2483007/range2483007/1

https://synbiohub.org/public/igem/BBa_K1982004/annotation2483007/range2483007/1
type: Range
persistent identity: range2483007
display id: range2483007
version: 1
direction: direction/reverse
top level: BBa_K1982004
start: 4123
end: 4668
orientation: inline

HA
type: SequenceAnnotation
persistent identity: annotation2517190
display id: annotation2517190
version: 1
top level: BBa_K1982004
role: SO:0000324
role: feature/tag
location: https://synbiohub.org/public/igem/BBa_K1982004/annotation2517190/range2517190/1

https://synbiohub.org/public/igem/BBa_K1982004/annotation2517190/range2517190/1
type: Range
persistent identity: range2517190
display id: range2517190
version: 1
direction: direction/forward
top level: BBa_K1982004
start: 4669
end: 4725
orientation: inline

ATG
type: SequenceAnnotation
persistent identity: annotation2483003
display id: annotation2483003
version: 1
top level: BBa_K1982004
role: SO:0000318
role: feature/start
location: https://synbiohub.org/public/igem/BBa_K1982004/annotation2483003/range2483003/1

https://synbiohub.org/public/igem/BBa_K1982004/annotation2483003/range2483003/1
type: Range
persistent identity: range2483003
display id: range2483003
version: 1
direction: direction/forward
top level: BBa_K1982004
start: 19
end: 21
orientation: inline

Prokaryotic rbs
type: SequenceAnnotation
persistent identity: annotation2517191
display id: annotation2517191
version: 1
top level: BBa_K1982004
role: feature/rbs
role: SO:0000139
location: https://synbiohub.org/public/igem/BBa_K1982004/annotation2517191/range2517191/1

https://synbiohub.org/public/igem/BBa_K1982004/annotation2517191/range2517191/1
type: Range
persistent identity: range2517191
display id: range2517191
version: 1
direction: direction/forward
top level: BBa_K1982004
start: 1
end: 18
orientation: inline

tCas9
type: SequenceAnnotation
persistent identity: annotation2483004
display id: annotation2483004
version: 1
top level: BBa_K1982004
role: SO:0000316
role: feature/protein
location: https://synbiohub.org/public/igem/BBa_K1982004/annotation2483004/range2483004/1

https://synbiohub.org/public/igem/BBa_K1982004/annotation2483004/range2483004/1
type: Range
persistent identity: range2483004
display id: range2483004
version: 1
direction: direction/reverse
top level: BBa_K1982004
start: 22
end: 4122
orientation: inline

TAATAA
type: SequenceAnnotation
persistent identity: annotation2483006
display id: annotation2483006
version: 1
top level: BBa_K1982004
role: SO:0000319
role: feature/stop
location: https://synbiohub.org/public/igem/BBa_K1982004/annotation2483006/range2483006/1

https://synbiohub.org/public/igem/BBa_K1982004/annotation2483006/range2483006/1
type: Range
persistent identity: range2483006
display id: range2483006
version: 1
direction: direction/forward
top level: BBa_K1982004
start: 4726
end: 4731
orientation: inline

BBa_K1982011
type: ComponentDefinition
persistent identity: BBa K1982011
display id: BBa_K1982011
version: 1
was generated by: https://synbiohub.org/public/igem/igem2sbol/1
description: tCas9-Vp64(Eukaryoticc)
creator: Zexu Li
created: 2016-10-08T11:00:00Z
modified: 2016-10-18T10:57:53Z
discontinued: false
dominant: false
group u list: _2449_
m user id: 30176
owner id: 30176
owning group id: 9
status: status/Planning
bookmark: false
mutable 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''' |[http://parts.igem.org/Help:Assembly_standard_10 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. 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). <!-- Add more about the biology of this part here ===Usage and Biology=== Protein data table for BioBrick <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_ ">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 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYL…
mutable notes: mutant Pst 1 :gctacctgcaG to gctacctgcaC mutant Pst 1 :actatctgcaG to actatctgcaC
mutable provenance: tCas9 is from GE Share company. VP64 is synthetic construct.
owned by: james
owned by: myers
star: false
type: DnaRegion
role: partType/Coding
role: SO:0000316
sequence annotation: Eukaryotic rbs
sequence annotation: FLAG
sequence annotation: VP16
sequence annotation: TAATAA
sequence annotation: HA
sequence annotation: ATG
sequence annotation: SV40 NLS
sequence annotation: tCas9
sequence annotation: Linker
sequence: https://synbiohub.org/public/igem/BBa_K1982011_sequence/1

Eukaryotic rbs
type: SequenceAnnotation
persistent identity: annotation2517313
display id: annotation2517313
version: 1
top level: BBa_K1982011
role: feature/rbs
role: SO:0000139
location: https://synbiohub.org/public/igem/BBa_K1982011/annotation2517313/range2517313/1

https://synbiohub.org/public/igem/BBa_K1982011/annotation2517313/range2517313/1
type: Range
persistent identity: range2517313
display id: range2517313
version: 1
direction: direction/forward
top level: BBa_K1982011
start: 1
end: 18
orientation: inline

FLAG
type: SequenceAnnotation
persistent identity: annotation2517323
display id: annotation2517323
version: 1
top level: BBa_K1982011
role: SO:0000324
role: feature/tag
location: https://synbiohub.org/public/igem/BBa_K1982011/annotation2517323/range2517323/1

https://synbiohub.org/public/igem/BBa_K1982011/annotation2517323/range2517323/1
type: Range
persistent identity: range2517323
display id: range2517323
version: 1
direction: direction/forward
top level: BBa_K1982011
start: 4339
end: 4362
orientation: inline

VP16
type: SequenceAnnotation
persistent identity: annotation2517318
display id: annotation2517318
version: 1
top level: BBa_K1982011
role: SO:0000110
role: feature/misc
location: https://synbiohub.org/public/igem/BBa_K1982011/annotation2517318/range2517318/1

https://synbiohub.org/public/igem/BBa_K1982011/annotation2517318/range2517318/1
type: Range
persistent identity: range2517318
display id: range2517318
version: 1
direction: direction/forward
top level: BBa_K1982011
start: 4183
end: 4221
orientation: inline

VP16
type: SequenceAnnotation
persistent identity: annotation2517319
display id: annotation2517319
version: 1
top level: BBa_K1982011
role: SO:0000110
role: feature/misc
location: https://synbiohub.org/public/igem/BBa_K1982011/annotation2517319/range2517319/1

https://synbiohub.org/public/igem/BBa_K1982011/annotation2517319/range2517319/1
type: Range
persistent identity: range2517319
display id: range2517319
version: 1
direction: direction/forward
top level: BBa_K1982011
start: 4222
end: 4260
orientation: inline

VP16
type: SequenceAnnotation
persistent identity: annotation2517317
display id: annotation2517317
version: 1
top level: BBa_K1982011
role: SO:0000110
role: feature/misc
location: https://synbiohub.org/public/igem/BBa_K1982011/annotation2517317/range2517317/1

https://synbiohub.org/public/igem/BBa_K1982011/annotation2517317/range2517317/1
type: Range
persistent identity: range2517317
display id: range2517317
version: 1
direction: direction/forward
top level: BBa_K1982011
start: 4144
end: 4182
orientation: inline

TAATAA
type: SequenceAnnotation
persistent identity: annotation2517324
display id: annotation2517324
version: 1
top level: BBa_K1982011
role: SO:0000319
role: feature/stop
location: https://synbiohub.org/public/igem/BBa_K1982011/annotation2517324/range2517324/1

https://synbiohub.org/public/igem/BBa_K1982011/annotation2517324/range2517324/1
type: Range
persistent identity: range2517324
display id: range2517324
version: 1
direction: direction/forward
top level: BBa_K1982011
start: 4363
end: 4368
orientation: inline

VP16
type: SequenceAnnotation
persistent identity: annotation2517320
display id: annotation2517320
version: 1
top level: BBa_K1982011
role: SO:0000110
role: feature/misc
location: https://synbiohub.org/public/igem/BBa_K1982011/annotation2517320/range2517320/1

https://synbiohub.org/public/igem/BBa_K1982011/annotation2517320/range2517320/1
type: Range
persistent identity: range2517320
display id: range2517320
version: 1
direction: direction/forward
top level: BBa_K1982011
start: 4261
end: 4299
orientation: inline

HA
type: SequenceAnnotation
persistent identity: annotation2517322
display id: annotation2517322
version: 1
top level: BBa_K1982011
role: SO:0000324
role: feature/tag
location: https://synbiohub.org/public/igem/BBa_K1982011/annotation2517322/range2517322/1

https://synbiohub.org/public/igem/BBa_K1982011/annotation2517322/range2517322/1
type: Range
persistent identity: range2517322
display id: range2517322
version: 1
direction: direction/forward
top level: BBa_K1982011
start: 4306
end: 4332
orientation: inline

ATG
type: SequenceAnnotation
persistent identity: annotation2517314
display id: annotation2517314
version: 1
top level: BBa_K1982011
role: SO:0000318
role: feature/start
location: https://synbiohub.org/public/igem/BBa_K1982011/annotation2517314/range2517314/1

https://synbiohub.org/public/igem/BBa_K1982011/annotation2517314/range2517314/1
type: Range
persistent identity: range2517314
display id: range2517314
version: 1
direction: direction/forward
top level: BBa_K1982011
start: 19
end: 21
orientation: inline

SV40 NLS
type: SequenceAnnotation
persistent identity: annotation2517316
display id: annotation2517316
version: 1
top level: BBa_K1982011
role: SO:0000110
role: feature/misc
location: https://synbiohub.org/public/igem/BBa_K1982011/annotation2517316/range2517316/1

https://synbiohub.org/public/igem/BBa_K1982011/annotation2517316/range2517316/1
type: Range
persistent identity: range2517316
display id: range2517316
version: 1
direction: direction/forward
top level: BBa_K1982011
start: 4123
end: 4143
orientation: inline

tCas9
type: SequenceAnnotation
persistent identity: annotation2517315
display id: annotation2517315
version: 1
top level: BBa_K1982011
role: SO:0000316
role: feature/protein
location: https://synbiohub.org/public/igem/BBa_K1982011/annotation2517315/range2517315/1

https://synbiohub.org/public/igem/BBa_K1982011/annotation2517315/range2517315/1
type: Range
persistent identity: range2517315
display id: range2517315
version: 1
direction: direction/forward
top level: BBa_K1982011
start: 22
end: 4122
orientation: inline

Linker
type: SequenceAnnotation
persistent identity: annotation2517321
display id: annotation2517321
version: 1
top level: BBa_K1982011
role: SO:0000110
role: feature/misc
location: https://synbiohub.org/public/igem/BBa_K1982011/annotation2517321/range2517321/1

https://synbiohub.org/public/igem/BBa_K1982011/annotation2517321/range2517321/1
type: Range
persistent identity: range2517321
display id: range2517321
version: 1
direction: direction/forward
top level: BBa_K1982011
start: 4300
end: 4305
orientation: inline

BBa_K2017000
type: ComponentDefinition
persistent identity: BBa K2017000
display id: BBa_K2017000
version: 1
was generated by: https://synbiohub.org/public/igem/igem2sbol/1
description: C-split Cas9 + DnaE C-intein
creator: Monica Victoria Gutierrez Salazar
created: 2016-10-02T11:00:00Z
modified: 2016-10-23T08:31:25Z
discontinued: false
dominant: false
experience: experience/Works
group u list: _2484_
m user id: 26626
owner id: 26626
owning group id: 9
status: status/Planning
bookmark: true
mutable description: Half of the SpCas9 protein fused to DnaE C-intein. The active full-lenght spCas9 can be recovered by using the other half of the protein, N-split Cas9 + DnaE N-intein. Inteins are used to fuse both parts of the protein, as they interact and splice, leaving the active protein.
mutable notes: As the part needs to be inserted in a viral vector, the overhangs have to be designed accordingly to the viral vector that is going to be used. The synthesis of C-split Cas9 could not be made in one gBlock, as it is too big. Hence, the half protein had to be splitted in two other parts for its synthesis.
mutable provenance: The protein domain is synthesized with the intein.
owned by: james
owned by: myers
star: false
type: DnaRegion
role: SO:0000417
role: partType/Protein Domain
sequence annotation: DnaE C-intein
sequence annotation: C-split Cas9
sequence: https://synbiohub.org/public/igem/BBa_K2017000_sequence/1

DnaE C-intein
type: SequenceAnnotation
persistent identity: annotation2486018
display id: annotation2486018
version: 1
top level: BBa_K2017000
role: SO:0000110
role: feature/misc
location: https://synbiohub.org/public/igem/BBa_K2017000/annotation2486018/range2486018/1

https://synbiohub.org/public/igem/BBa_K2017000/annotation2486018/range2486018/1
type: Range
persistent identity: range2486018
display id: range2486018
version: 1
direction: direction/forward
top level: BBa_K2017000
start: 1
end: 108
orientation: inline

C-split Cas9
type: SequenceAnnotation
persistent identity: annotation2486019
display id: annotation2486019
version: 1
top level: BBa_K2017000
role: SO:0000316
role: feature/protein
location: https://synbiohub.org/public/igem/BBa_K2017000/annotation2486019/range2486019/1

https://synbiohub.org/public/igem/BBa_K2017000/annotation2486019/range2486019/1
type: Range
persistent identity: range2486019
display id: range2486019
version: 1
direction: direction/forward
top level: BBa_K2017000
start: 109
end: 2358
orientation: inline

TC
type: ComponentDefinition
persistent identity: BBa K1982000
display id: BBa_K1982000
version: 1
was generated by: https://synbiohub.org/public/igem/igem2sbol/1
description: tCas9-CIBN (Prokaryotic LACE system)
creator: Zexu Li
created: 2016-09-09T11:00:00Z
modified: 2016-10-18T10:12:41Z
discontinued: false
dominant: false
experience: experience/Works
group u list: _2449_
m user id: 30176
owner id: 30176
owning group id: 9
status: status/Planning
bookmark: false
mutable description: tCas9 is a standardized (RFC 10) protein.Interacting with a DNA-binding RNA and fused with different effector domains it can be used for specific gene regulation. Cas9 is the main protein of the CRISPR/Cas system II of Streptococcus pyogenes. CRISPR systems protect bacteria and archaea from phages by recognizing and cleaving of invading phage DNA.This recognition is based on Watson Crick base pairing between a short RNA, called crRNA, and the complementary DNA strand. A second RNA, called tracrRNA, connects crRNA and Cas9. These three parts together form a protein-RNA-DNA complex with the targeted DNA strand [1] Cas9 became of great interest for research concerning DNA targeting, because of its ability to recognize site specific DNA strands by a crRNA. At first the functionality of Cas9 was modified by exchanging aminoacids. As a result, Cas9 was able to introduce mutations within the genome of several organisms by causing double strand breaks [2][3]. Then, it was converted from a nuclease to a nickase introducing single strand breaks [4] and lately it was converted to an enzymatically inactive form, called dCas9 [5]. This tCas9 is standardized (RFC 10). It can be used as a DNA binding protein, that can be fused with different effectors in order to regulate gene expression.
mutable notes: mutant Pst 1 :gctacctgcaG to gctacctgcaC mutant Pst 1 :actatctgcaG to actatctgcaC
mutable provenance: GE Share
owned by: james
owned by: myers
star: false
type: DnaRegion
role: partType/Device
role: SO:0000804
sequence annotation: tCas9
sequence annotation: HA
sequence annotation: Prokaryotic rbs
sequence annotation: TAATAA
sequence annotation: ATG
sequence annotation: CIBN
sequence: https://synbiohub.org/public/igem/BBa_K1982000_sequence/1

tCas9
type: SequenceAnnotation
persistent identity: annotation2517189
display id: annotation2517189
version: 1
top level: TC
role: SO:0000316
role: feature/protein
location: https://synbiohub.org/public/igem/BBa_K1982000/annotation2517189/range2517189/1

https://synbiohub.org/public/igem/BBa_K1982000/annotation2517189/range2517189/1
type: Range
persistent identity: range2517189
display id: range2517189
version: 1
direction: direction/forward
top level: TC
start: 22
end: 4122
orientation: inline

HA
type: SequenceAnnotation
persistent identity: annotation2517186
display id: annotation2517186
version: 1
top level: TC
role: SO:0000324
role: feature/tag
location: https://synbiohub.org/public/igem/BBa_K1982000/annotation2517186/range2517186/1

https://synbiohub.org/public/igem/BBa_K1982000/annotation2517186/range2517186/1
type: Range
persistent identity: range2517186
display id: range2517186
version: 1
direction: direction/forward
top level: TC
start: 4669
end: 4725
orientation: inline

Prokaryotic rbs
type: SequenceAnnotation
persistent identity: annotation2517184
display id: annotation2517184
version: 1
top level: TC
role: feature/rbs
role: SO:0000139
location: https://synbiohub.org/public/igem/BBa_K1982000/annotation2517184/range2517184/1

https://synbiohub.org/public/igem/BBa_K1982000/annotation2517184/range2517184/1
type: Range
persistent identity: range2517184
display id: range2517184
version: 1
direction: direction/forward
top level: TC
start: 1
end: 18
orientation: inline

TAATAA
type: SequenceAnnotation
persistent identity: annotation2517187
display id: annotation2517187
version: 1
top level: TC
role: SO:0000319
role: feature/stop
location: https://synbiohub.org/public/igem/BBa_K1982000/annotation2517187/range2517187/1

https://synbiohub.org/public/igem/BBa_K1982000/annotation2517187/range2517187/1
type: Range
persistent identity: range2517187
display id: range2517187
version: 1
direction: direction/forward
top level: TC
start: 4726
end: 4731
orientation: inline

ATG
type: SequenceAnnotation
persistent identity: annotation2517185
display id: annotation2517185
version: 1
top level: TC
role: SO:0000318
role: feature/start
location: https://synbiohub.org/public/igem/BBa_K1982000/annotation2517185/range2517185/1

https://synbiohub.org/public/igem/BBa_K1982000/annotation2517185/range2517185/1
type: Range
persistent identity: range2517185
display id: range2517185
version: 1
direction: direction/forward
top level: TC
start: 19
end: 21
orientation: inline

CIBN
type: SequenceAnnotation
persistent identity: annotation2517188
display id: annotation2517188
version: 1
top level: TC
role: SO:0000316
role: feature/protein
location: https://synbiohub.org/public/igem/BBa_K1982000/annotation2517188/range2517188/1

https://synbiohub.org/public/igem/BBa_K1982000/annotation2517188/range2517188/1
type: Range
persistent identity: range2517188
display id: range2517188
version: 1
direction: direction/forward
top level: TC
start: 4123
end: 4668
orientation: inline

BBa_K1559005
type: ComponentDefinition
persistent identity: BBa K1559005
display id: BBa_K1559005
version: 1
was generated by: https://synbiohub.org/public/igem/igem2sbol/1
description: CRISPR/Cas9 system with Anderson low-expression constitutive promoter
creator: Xiuqi (Rex) Xia
created: 2014-10-12T11:00:00Z
modified: 2015-05-08T01:10:53Z
discontinued: false
dominant: false
group u list: _1944_
m user id: 0
owner id: 23047
owning group id: 9
sample status: Not in stock
status: status/Unavailable
bookmark: false
mutable description: TODO
mutable notes: TODO
mutable provenance: TODO
owned by: james
owned by: myers
star: false
type: DnaRegion
role: SO:0000804
role: partType/Generator
component: BBa_K1559002
component: BBa_B0034
component: BBa_J23114
sequence annotation: BBa_K1559002
sequence annotation: BBa_J23114
sequence annotation: BBa_B0034
sequence: https://synbiohub.org/public/igem/BBa_K1559005_sequence/1

BBa_K1559002
type: Component
persistent identity: component2418649
display id: component2418649
version: 1
top level: BBa_K1559005
access: public
definition: BBa_K1559002

BBa_B0034
type: Component
persistent identity: component2418648
display id: component2418648
version: 1
top level: BBa_K1559005
access: public
definition: https://synbiohub.org/public/igem/BBa_B0034/1

BBa_J23114
type: Component
persistent identity: component2418646
display id: component2418646
version: 1
top level: BBa_K1559005
access: public
definition: https://synbiohub.org/public/igem/BBa_J23114/1

BBa_K1559002
type: SequenceAnnotation
persistent identity: annotation2418649
display id: annotation2418649
version: 1
top level: BBa_K1559005
location: https://synbiohub.org/public/igem/BBa_K1559005/annotation2418649/range2418649/1
component: BBa_K1559002

https://synbiohub.org/public/igem/BBa_K1559005/annotation2418649/range2418649/1
type: Range
persistent identity: range2418649
display id: range2418649
version: 1
direction: direction/reverse
top level: BBa_K1559005
start: 48
end: 5127
orientation: inline

BBa_J23114
type: SequenceAnnotation
persistent identity: annotation2418646
display id: annotation2418646
version: 1
top level: BBa_K1559005
location: https://synbiohub.org/public/igem/BBa_K1559005/annotation2418646/range2418646/1
component: BBa_J23114

https://synbiohub.org/public/igem/BBa_K1559005/annotation2418646/range2418646/1
type: Range
persistent identity: range2418646
display id: range2418646
version: 1
direction: direction/reverse
top level: BBa_K1559005
start: 1
end: 35
orientation: inline

BBa_B0034
type: SequenceAnnotation
persistent identity: annotation2418648
display id: annotation2418648
version: 1
top level: BBa_K1559005
location: https://synbiohub.org/public/igem/BBa_K1559005/annotation2418648/range2418648/1
component: BBa_B0034

https://synbiohub.org/public/igem/BBa_K1559005/annotation2418648/range2418648/1
type: Range
persistent identity: range2418648
display id: range2418648
version: 1
direction: direction/reverse
top level: BBa_K1559005
start: 36
end: 47
orientation: inline

BBa_K2017013
type: ComponentDefinition
persistent identity: BBa K2017013
display id: BBa_K2017013
version: 1
was generated by: https://synbiohub.org/public/igem/igem2sbol/1
description: 35s + TFL consense + AEK-Luciferase + Tnos
creator: Monica Victoria Gutierrez Salazar
created: 2016-10-03T11:00:00Z
modified: 2016-10-22T05:16:14Z
discontinued: false
dominant: false
group u list: _2484_
m user id: 26626
owner id: 26626
owning group id: 9
status: status/Planning
bookmark: false
mutable description: Modul of the gRNA testing system created by Valencia UPV 2016 team. This system aims to test the functionality and efficiency of a chosen gRNA to target a particular gene. This system is modular, as the gene inserted can be chosen by the user, only taking into account the required overhangs (5'-AATG-3' and 5'-TTCG-3'). It includes a promoter 35s (BBa_K1537015), 20 nucleotides of the TFL consensus gene of Clemenules (clementine orange), the reporter luciferase with AEK linker and the terminator Tnos. Luciferase is out of its reading frame due to a nucleotide added upstream to the linker, and the initial ATG has been removed. This means that when the luciferase is translated, it will not be functional. To make it functional, it is necessary to make indels in the TFL fragment that put the luciferase in the correct reading frame. These indels can be made using the CRISPR/Cas9 system, which is the aim of this device.
mutable notes: It is important to notice that this device is modular. To insert the region of the gene of interest, this region must have the overhang 5'-AATG-3' and 5'-TTCG-3'.
mutable provenance: This part was obtained using GoldenBraid assembly. The TFL consense sequence was obtained with oligonucleotide synthesis.
owned by: james
owned by: myers
star: false
type: DnaRegion
role: partType/Device
role: SO:0000804
component: BBa_K1484215
component: BBa_K1537015
component: BBa_K2017005
sequence annotation: BBa_K1537015
sequence annotation: BBa_K1484215
sequence annotation: BBa_K2017005
sequence: https://synbiohub.org/public/igem/BBa_K2017013_sequence/1

BBa_K1484215
type: Component
persistent identity: component2487464
display id: component2487464
version: 1
top level: BBa_K2017013
access: public
definition: https://synbiohub.org/public/igem/BBa_K1484215/1

BBa_K1537015
type: Component
persistent identity: component2487457
display id: component2487457
version: 1
top level: BBa_K2017013
access: public
definition: https://synbiohub.org/public/igem/BBa_K1537015/1

BBa_K2017005
type: Component
persistent identity: component2487461
display id: component2487461
version: 1
top level: BBa_K2017013
access: public
definition: https://synbiohub.org/public/igem/BBa_K2017005/1

BBa_K1537015
type: SequenceAnnotation
persistent identity: annotation2487457
display id: annotation2487457
version: 1
top level: BBa_K2017013
location: https://synbiohub.org/public/igem/BBa_K2017013/annotation2487457/range2487457/1
component: BBa_K1537015

https://synbiohub.org/public/igem/BBa_K2017013/annotation2487457/range2487457/1
type: Range
persistent identity: range2487457
display id: range2487457
version: 1
direction: direction/reverse
top level: BBa_K2017013
start: 1
end: 860
orientation: inline

BBa_K1484215
type: SequenceAnnotation
persistent identity: annotation2487464
display id: annotation2487464
version: 1
top level: BBa_K2017013
location: https://synbiohub.org/public/igem/BBa_K2017013/annotation2487464/range2487464/1
component: BBa_K1484215

https://synbiohub.org/public/igem/BBa_K2017013/annotation2487464/range2487464/1
type: Range
persistent identity: range2487464
display id: range2487464
version: 1
direction: direction/reverse
top level: BBa_K2017013
start: 2580
end: 2872
orientation: inline

BBa_K2017005
type: SequenceAnnotation
persistent identity: annotation2487461
display id: annotation2487461
version: 1
top level: BBa_K2017013
location: https://synbiohub.org/public/igem/BBa_K2017013/annotation2487461/range2487461/1
component: BBa_K2017005

https://synbiohub.org/public/igem/BBa_K2017013/annotation2487461/range2487461/1
type: Range
persistent identity: range2487461
display id: range2487461
version: 1
direction: direction/reverse
top level: BBa_K2017013
start: 869
end: 2571
orientation: inline

BBa_K1137014
type: ComponentDefinition
persistent identity: BBa K1137014
display id: BBa_K1137014
version: 1
was generated by: https://synbiohub.org/public/igem/igem2sbol/1
description: tracRNA-CAS9
creator: Nicolas Koutsoubelis, Anne L??chner
created: 2013-10-03T11:00:00Z
modified: 2015-05-08T01:09:21Z
discontinued: false
dominant: false
experience: experience/None
group u list: _1449_
m user id: 0
owner id: 11926
owning group id: 9
sample status: Not in stock
status: status/Unavailable
bookmark: false
mutable description: This complex part contains the tracrRNA and the CAS9 under the control of constitutive promoters. Note that there is no RBS before the tracRNA. The tracrRNA-CAS9 part is the heart of the CRISPR system. The tracrRNA and the corresponding target crRNA dimerize and are processed and hybridize before binding to the CAS9 protein. The tracrRNA has no specific part that is responsible for binding to the targeted DNA. Therefor this part can be used together with any crRNA. The CAS9 gene encodes for the CRISPR associated protein 9 that is needed to generate a double strand break at a target site. CAS9 binds to the tracRNA-crRNA dimer/gRNA and is then guided by them/it to the target site where it generate a double strand break. This part can be used together with the crRNA anti KAN or the gRNA anti KAN and cause double strand breaks. It can of course also be used together with new generated crRNAs/gRNAs. The tracRNA-CAS9 part is taken from the CRISPER plasmid of the Jiang et al. 2013 paper.
mutable notes: -
mutable provenance: The plasmid was ordered from Addgene and come originally from the paper below: Wenyan Jiang, David Bikard, David Cox, Feng Zhang & Luciano A Marraffini (2013): RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nature Biotechnology 31 (3), 233-239.
owned by: james
owned by: myers
star: false
type: DnaRegion
role: partType/Coding
role: SO:0000316
sequence: https://synbiohub.org/public/igem/BBa_K1137014_sequence/1

BBa_K1982002
type: ComponentDefinition
persistent identity: BBa K1982002
display id: BBa_K1982002
version: 1
was generated by: https://synbiohub.org/public/igem/igem2sbol/1
description: Prokaryotic Cryptochrome 2 (CRY2) ( a blue light stimulated photoreceptor)
creator: Zexu Li
created: 2016-09-10T11:00:00Z
modified: 2016-10-09T12:15:31Z
discontinued: false
dominant: false
experience: experience/Works
group u list: _2449_
m user id: 30176
owner id: 30176
owning group id: 9
status: status/Planning
bookmark: false
mutable description: Cryptochrome 2 (CRY2) is a blue light stimulated photoreceptor, when exposed to blue light, it would interact withthe N-terminal fragment of CIB1 (CIBN) . This part is full-length CRY2 CDS. Optogenetic systems enable precise spatial and temporal control of cell behavior. A light-activated CRISPR/Cas9 effector (LACE) system that induces transcription of endogenous genes in the presence of blue light.This was accomplished by fusing the light-inducible heterodimerizing proteins CRY2 and CIB1 to a transactivation domain and the catalytically inactive tCas9, respectively. The versatile LACE system can be easily directed to new DNA sequences for the dynamic regulation of endogenous genes.
mutable notes: mutant EcoR 1 : TGTTGGGAATTc--->TGTTGGGAATTg
mutable provenance: The light-inducible heterodimerizering proteins CRY2 and CIB1 are both from Arabidopsis thaliana.
owned by: james
owned by: myers
star: false
type: DnaRegion
role: partType/Coding
role: SO:0000316
sequence annotation: ATG
sequence annotation: Prokaryotic rbs
sequence: https://synbiohub.org/public/igem/BBa_K1982002_sequence/1

ATG
type: SequenceAnnotation
persistent identity: annotation2483021
display id: annotation2483021
version: 1
top level: BBa_K1982002
role: SO:0000318
role: feature/start
location: https://synbiohub.org/public/igem/BBa_K1982002/annotation2483021/range2483021/1

https://synbiohub.org/public/igem/BBa_K1982002/annotation2483021/range2483021/1
type: Range
persistent identity: range2483021
display id: range2483021
version: 1
direction: direction/forward
top level: BBa_K1982002
start: 19
end: 21
orientation: inline

Prokaryotic rbs
type: SequenceAnnotation
persistent identity: annotation2488670
display id: annotation2488670
version: 1
top level: BBa_K1982002
role: feature/rbs
role: SO:0000139
location: https://synbiohub.org/public/igem/BBa_K1982002/annotation2488670/range2488670/1

https://synbiohub.org/public/igem/BBa_K1982002/annotation2488670/range2488670/1
type: Range
persistent identity: range2488670
display id: range2488670
version: 1
direction: direction/forward
top level: BBa_K1982002
start: 1
end: 18
orientation: inline

BBa_K2017010
type: ComponentDefinition
persistent identity: BBa K2017010
display id: BBa_K2017010
version: 1
was generated by: https://synbiohub.org/public/igem/igem2sbol/1
description: 35s + Ga20ox consense + RSIAT-TEV-Luciferase + Tnos
creator: Monica Victoria Gutierrez Salazar
created: 2016-10-03T11:00:00Z
modified: 2016-10-22T05:13:20Z
discontinued: false
dominant: false
group u list: _2484_
m user id: 26626
owner id: 26626
owning group id: 9
status: status/Planning
bookmark: false
mutable description: Modul of the gRNA testing system created by Valencia UPV 2016 team. This system aims to test the functionality and efficiency of a chosen gRNA to target a particular gene. This system is modular, as the gene inserted can be chosen by the user, only taking into account the required overhangs (5'-AATG-3' and 5'-TTCG-3'). It includes a promoter 35s (BBa_K1537015), 20 nucleotides of the Ga20ox consensus gene of Oryza sativa (rice), the reporter luciferase with RSIAT+TEV linker and the terminator Tnos. Luciferase is out of its reading frame due to a nucleotide added upstream to the linker, and the initial ATG has been removed. This means that when the luciferase is translated, it will not be functional. To make it functional, it is necessary to make indels in the Ga20ox fragment that put the luciferase in the correct reading frame. These indels can be made using the CRISPR/Cas9 system, which is the aim of this device.
mutable notes: It is important to notice that this device is modular. To insert the region of the gene of interest, this region must have the overhang 5'-AATG-3' and 5'-TTCG-3'.
mutable provenance: This part was obtained using GoldenBraid assembly. The Ga20ox consense sequence was obtained with oligonucleotide synthesis.
owned by: james
owned by: myers
star: false
type: DnaRegion
role: partType/Device
role: SO:0000804
component: BBa_K2017006
component: BBa_K1537015
component: BBa_K1484215
sequence annotation: BBa_K2017006
sequence annotation: BBa_K1484215
sequence annotation: BBa_K1537015
sequence: https://synbiohub.org/public/igem/BBa_K2017010_sequence/1

BBa_K2017006
type: Component
persistent identity: component2487430
display id: component2487430
version: 1
top level: BBa_K2017010
access: public
definition: https://synbiohub.org/public/igem/BBa_K2017006/1

BBa_K1537015
type: Component
persistent identity: component2487426
display id: component2487426
version: 1
top level: BBa_K2017010
access: public
definition: https://synbiohub.org/public/igem/BBa_K1537015/1

BBa_K1484215
type: Component
persistent identity: component2487433
display id: component2487433
version: 1
top level: BBa_K2017010
access: public
definition: https://synbiohub.org/public/igem/BBa_K1484215/1

BBa_K2017006
type: SequenceAnnotation
persistent identity: annotation2487430
display id: annotation2487430
version: 1
top level: BBa_K2017010
location: https://synbiohub.org/public/igem/BBa_K2017010/annotation2487430/range2487430/1
component: BBa_K2017006

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type: Range
persistent identity: range2487430
display id: range2487430
version: 1
direction: direction/reverse
top level: BBa_K2017010
start: 869
end: 2568
orientation: inline

BBa_K1484215
type: SequenceAnnotation
persistent identity: annotation2487433
display id: annotation2487433
version: 1
top level: BBa_K2017010
location: https://synbiohub.org/public/igem/BBa_K2017010/annotation2487433/range2487433/1
component: BBa_K1484215

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type: Range
persistent identity: range2487433
display id: range2487433
version: 1
direction: direction/reverse
top level: BBa_K2017010
start: 2577
end: 2869
orientation: inline

BBa_K1537015
type: SequenceAnnotation
persistent identity: annotation2487426
display id: annotation2487426
version: 1
top level: BBa_K2017010
location: https://synbiohub.org/public/igem/BBa_K2017010/annotation2487426/range2487426/1
component: BBa_K1537015

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type: Range
persistent identity: range2487426
display id: range2487426
version: 1
direction: direction/reverse
top level: BBa_K2017010
start: 1
end: 860
orientation: inline

BBa_K1982003
type: ComponentDefinition
persistent identity: BBa K1982003
display id: BBa_K1982003
version: 1
was generated by: https://synbiohub.org/public/igem/igem2sbol/1
description: CIBN(the N-terminal fragment of CIB1)
creator: Zexu Li
created: 2016-09-11T11:00:00Z
modified: 2016-09-12T05:35:21Z
discontinued: false
dominant: false
experience: experience/Works
group u list: _2449_
m user id: 30176
owner id: 30176
owning group id: 9
status: status/Planning
bookmark: false
mutable description: Encodes the N-terminal fragment of transcription factor CIB1 (cryptochrome-interacting basic-helix-loop-helix). CIB1 interacts with CRY2 (cryptochrome 2) in a blue light-specific manner in yeast and Arabidopsis cells, and it acts together with additional CIB1-related proteins to promote CRY2-dependent floral initiation. CIB1 positively regulates FT expression. Cryptochrome 2 (CRY2) is a blue light stimulated photoreceptor, when exposed to blue light, it would interact withthe N-terminal fragment of CIB1 (CIBN) . Optogenetic systems enable precise spatial and temporal control of cell behavior. A light-activated CRISPR/Cas9 effector (LACE) system that induces transcription of endogenous genes in the presence of blue light.This was accomplished by fusing the light-inducible heterodimerizing proteins CRY2 and CIB1 to a transactivation domain and the catalytically inactive tCas9, respectively. The versatile LACE system can be easily directed to new DNA sequences for the dynamic regulation of endogenous genes[1].
mutable notes: no
mutable provenance: The light-inducible heterodimerizering proteins CRY2 and CIB1 are both from Arabidopsis thaliana.
owned by: james
owned by: myers
star: false
type: DnaRegion
role: partType/Coding
role: SO:0000316
sequence annotation: TAA
sequence annotation: ATG
sequence: https://synbiohub.org/public/igem/BBa_K1982003_sequence/1

TAA
type: SequenceAnnotation
persistent identity: annotation2482939
display id: annotation2482939
version: 1
top level: BBa_K1982003
role: SO:0000319
role: feature/stop
location: https://synbiohub.org/public/igem/BBa_K1982003/annotation2482939/range2482939/1

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type: Range
persistent identity: range2482939
display id: range2482939
version: 1
direction: direction/forward
top level: BBa_K1982003
start: 607
end: 612
orientation: inline

ATG
type: SequenceAnnotation
persistent identity: annotation2482938
display id: annotation2482938
version: 1
top level: BBa_K1982003
role: SO:0000318
role: feature/start
location: https://synbiohub.org/public/igem/BBa_K1982003/annotation2482938/range2482938/1

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type: Range
persistent identity: range2482938
display id: range2482938
version: 1
direction: direction/forward
top level: BBa_K1982003
start: 1
end: 3
orientation: inline

BBa_K1559002
type: ComponentDefinition
persistent identity: BBa K1559002
display id: BBa_K1559002
version: 1
was generated by: https://synbiohub.org/public/igem/igem2sbol/1
description: rearranged CRISPR/Cas9 system without promoter
creator: Xiuqi (Rex) Xia
created: 2014-10-09T11:00:00Z
modified: 2015-05-08T01:10:53Z
discontinued: false
dominant: false
experience: experience/None
group u list: _1944_
m user id: 0
owner id: 23047
owning group id: 9
sample status: Not in stock
status: status/Unavailable
bookmark: false
mutable description: TODO
mutable notes: TODO
mutable provenance: TODO
owned by: james
owned by: myers
star: false
type: DnaRegion
role: partType/Coding
role: SO:0000316
sequence annotation: direct repeat
sequence annotation: cas9
sequence annotation: crRNA array
sequence annotation: Region containing bidirectional constitutive promoter
sequence annotation: tracrRNA
sequence annotation: stem loop within tracrRNA
sequence annotation: predicted terminator fwd
sequence annotation: predicted terminator rev
sequence annotation: spacer placeholder containing BsaI sites
sequence: https://synbiohub.org/public/igem/BBa_K1559002_sequence/1

direct repeat
type: SequenceAnnotation
persistent identity: annotation2418814
display id: annotation2418814
version: 1
top level: BBa_K1559002
role: SO:0000110
role: feature/misc
location: https://synbiohub.org/public/igem/BBa_K1559002/annotation2418814/range2418814/1

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type: Range
persistent identity: range2418814
display id: range2418814
version: 1
direction: direction/forward
top level: BBa_K1559002
start: 4326
end: 4361
orientation: inline

cas9
type: SequenceAnnotation
persistent identity: annotation2418811
display id: annotation2418811
version: 1
top level: BBa_K1559002
role: feature/cds
role: SO:0000316
location: https://synbiohub.org/public/igem/BBa_K1559002/annotation2418811/range2418811/1

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type: Range
persistent identity: range2418811
display id: range2418811
version: 1
direction: direction/forward
top level: BBa_K1559002
start: 1
end: 4107
orientation: inline

crRNA array
type: SequenceAnnotation
persistent identity: annotation2418815
display id: annotation2418815
version: 1
top level: BBa_K1559002
role: SO:0000110
role: feature/misc
location: https://synbiohub.org/public/igem/BBa_K1559002/annotation2418815/range2418815/1

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type: Range
persistent identity: range2418815
display id: range2418815
version: 1
direction: direction/forward
top level: BBa_K1559002
start: 4260
end: 4361
orientation: inline

Region containing bidirectional constitutive promoter
type: SequenceAnnotation
persistent identity: annotation2418817
display id: annotation2418817
version: 1
top level: BBa_K1559002
role: SO:0000110
role: feature/misc
location: https://synbiohub.org/public/igem/BBa_K1559002/annotation2418817/range2418817/1

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type: Range
persistent identity: range2418817
display id: range2418817
version: 1
direction: direction/forward
top level: BBa_K1559002
start: 4871
end: 5080
orientation: inline

tracrRNA
type: SequenceAnnotation
persistent identity: annotation2418816
display id: annotation2418816
version: 1
top level: BBa_K1559002
role: SO:0000110
role: feature/misc
location: https://synbiohub.org/public/igem/BBa_K1559002/annotation2418816/range2418816/1

https://synbiohub.org/public/igem/BBa_K1559002/annotation2418816/range2418816/1
type: Range
persistent identity: range2418816
display id: range2418816
version: 1
direction: direction/reverse
top level: BBa_K1559002
start: 4700
end: 4870
orientation: inline

direct repeat
type: SequenceAnnotation
persistent identity: annotation2418812
display id: annotation2418812
version: 1
top level: BBa_K1559002
role: SO:0000110
role: feature/misc
location: https://synbiohub.org/public/igem/BBa_K1559002/annotation2418812/range2418812/1

https://synbiohub.org/public/igem/BBa_K1559002/annotation2418812/range2418812/1
type: Range
persistent identity: range2418812
display id: range2418812
version: 1
direction: direction/reverse
top level: BBa_K1559002
start: 4260
end: 4295
orientation: inline

stem loop within tracrRNA
type: SequenceAnnotation
persistent identity: annotation2425040
display id: annotation2425040
version: 1
top level: BBa_K1559002
role: feature/stem loop
role: SO:0000313
location: https://synbiohub.org/public/igem/BBa_K1559002/annotation2425040/range2425040/1

https://synbiohub.org/public/igem/BBa_K1559002/annotation2425040/range2425040/1
type: Range
persistent identity: range2425040
display id: range2425040
version: 1
direction: direction/reverse
top level: BBa_K1559002
start: 4692
end: 4733
orientation: inline

predicted terminator fwd
type: SequenceAnnotation
persistent identity: annotation2425031
display id: annotation2425031
version: 1
top level: BBa_K1559002
role: feature/stem loop
role: SO:0000313
location: https://synbiohub.org/public/igem/BBa_K1559002/annotation2425031/range2425031/1

https://synbiohub.org/public/igem/BBa_K1559002/annotation2425031/range2425031/1
type: Range
persistent identity: range2425031
display id: range2425031
version: 1
direction: direction/forward
top level: BBa_K1559002
start: 4628
end: 4676
orientation: inline

predicted terminator rev
type: SequenceAnnotation
persistent identity: annotation2425010
display id: annotation2425010
version: 1
top level: BBa_K1559002
role: feature/stem loop
role: SO:0000313
location: https://synbiohub.org/public/igem/BBa_K1559002/annotation2425010/range2425010/1

https://synbiohub.org/public/igem/BBa_K1559002/annotation2425010/range2425010/1
type: Range
persistent identity: range2425010
display id: range2425010
version: 1
direction: direction/reverse
top level: BBa_K1559002
start: 4628
end: 4674
orientation: inline

spacer placeholder containing BsaI sites
type: SequenceAnnotation
persistent identity: annotation2418813
display id: annotation2418813
version: 1
top level: BBa_K1559002
role: SO:0000110
role: feature/misc
location: https://synbiohub.org/public/igem/BBa_K1559002/annotation2418813/range2418813/1

https://synbiohub.org/public/igem/BBa_K1559002/annotation2418813/range2418813/1
type: Range
persistent identity: range2418813
display id: range2418813
version: 1
direction: direction/forward
top level: BBa_K1559002
start: 4296
end: 4325
orientation: inline

BBa_K1559008
type: ComponentDefinition
persistent identity: BBa K1559008
display id: BBa_K1559008
version: 1
was generated by: https://synbiohub.org/public/igem/igem2sbol/1
description: CRISPR/Cas9 system with pRha inducible promoter
creator: Xiuqi (Rex) Xia
created: 2014-10-12T11:00:00Z
modified: 2015-05-08T01:10:53Z
discontinued: false
dominant: false
group u list: _1944_
m user id: 0
owner id: 23047
owning group id: 9
sample status: Not in stock
status: status/Unavailable
bookmark: false
mutable description: TODO
mutable notes: TODO
mutable provenance: TODO
owned by: james
owned by: myers
star: false
type: DnaRegion
role: SO:0000804
role: partType/Generator
component: BBa_B0034
component: BBa_K1559002
component: BBa_K914003
sequence annotation: BBa_K1559002
sequence annotation: BBa_K914003
sequence annotation: BBa_B0034
sequence: https://synbiohub.org/public/igem/BBa_K1559008_sequence/1

BBa_B0034
type: Component
persistent identity: component2418693
display id: component2418693
version: 1
top level: BBa_K1559008
access: public
definition: https://synbiohub.org/public/igem/BBa_B0034/1

BBa_K1559002
type: Component
persistent identity: component2418694
display id: component2418694
version: 1
top level: BBa_K1559008
access: public
definition: BBa_K1559002

BBa_K914003
type: Component
persistent identity: component2418691
display id: component2418691
version: 1
top level: BBa_K1559008
access: public
definition: https://synbiohub.org/public/igem/BBa_K914003/1

BBa_K1559002
type: SequenceAnnotation
persistent identity: annotation2418694
display id: annotation2418694
version: 1
top level: BBa_K1559008
location: https://synbiohub.org/public/igem/BBa_K1559008/annotation2418694/range2418694/1
component: BBa_K1559002

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type: Range
persistent identity: range2418694
display id: range2418694
version: 1
direction: direction/reverse
top level: BBa_K1559008
start: 135
end: 5214
orientation: inline

BBa_K914003
type: SequenceAnnotation
persistent identity: annotation2418691
display id: annotation2418691
version: 1
top level: BBa_K1559008
location: https://synbiohub.org/public/igem/BBa_K1559008/annotation2418691/range2418691/1
component: BBa_K914003

https://synbiohub.org/public/igem/BBa_K1559008/annotation2418691/range2418691/1
type: Range
persistent identity: range2418691
display id: range2418691
version: 1
direction: direction/reverse
top level: BBa_K1559008
start: 1
end: 122
orientation: inline

BBa_B0034
type: SequenceAnnotation
persistent identity: annotation2418693
display id: annotation2418693
version: 1
top level: BBa_K1559008
location: https://synbiohub.org/public/igem/BBa_K1559008/annotation2418693/range2418693/1
component: BBa_B0034

https://synbiohub.org/public/igem/BBa_K1559008/annotation2418693/range2418693/1
type: Range
persistent identity: range2418693
display id: range2418693
version: 1
direction: direction/reverse
top level: BBa_K1559008
start: 123
end: 134
orientation: inline

BBa_K1559003
type: ComponentDefinition
persistent identity: BBa K1559003
display id: BBa_K1559003
version: 1
was generated by: https://synbiohub.org/public/igem/igem2sbol/1
description: CRISPR/Cas9 system with Anderson high-expression constitutive promoter
creator: Xiuqi (Rex) Xia
created: 2014-10-12T11:00:00Z
modified: 2015-05-08T01:10:53Z
discontinued: false
dominant: false
group u list: _1944_
m user id: 0
owner id: 23047
owning group id: 9
sample status: Not in stock
status: status/Unavailable
bookmark: false
mutable description: TODO
mutable notes: TODO
mutable provenance: TODO
owned by: james
owned by: myers
star: false
type: DnaRegion
role: SO:0000804
role: partType/Generator
component: BBa_J23119
component: BBa_K1559002
component: BBa_B0034
sequence annotation: BBa_K1559002
sequence annotation: BBa_B0034
sequence annotation: BBa_J23119
sequence: https://synbiohub.org/public/igem/BBa_K1559003_sequence/1

BBa_J23119
type: Component
persistent identity: component2418638
display id: component2418638
version: 1
top level: BBa_K1559003
access: public
definition: https://synbiohub.org/public/igem/BBa_J23119/1

BBa_K1559002
type: Component
persistent identity: component2418641
display id: component2418641
version: 1
top level: BBa_K1559003
access: public
definition: BBa_K1559002

BBa_B0034
type: Component
persistent identity: component2418640
display id: component2418640
version: 1
top level: BBa_K1559003
access: public
definition: https://synbiohub.org/public/igem/BBa_B0034/1

BBa_K1559002
type: SequenceAnnotation
persistent identity: annotation2418641
display id: annotation2418641
version: 1
top level: BBa_K1559003
location: https://synbiohub.org/public/igem/BBa_K1559003/annotation2418641/range2418641/1
component: BBa_K1559002

https://synbiohub.org/public/igem/BBa_K1559003/annotation2418641/range2418641/1
type: Range
persistent identity: range2418641
display id: range2418641
version: 1
direction: direction/reverse
top level: BBa_K1559003
start: 48
end: 5127
orientation: inline

BBa_B0034
type: SequenceAnnotation
persistent identity: annotation2418640
display id: annotation2418640
version: 1
top level: BBa_K1559003
location: https://synbiohub.org/public/igem/BBa_K1559003/annotation2418640/range2418640/1
component: BBa_B0034

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type: Range
persistent identity: range2418640
display id: range2418640
version: 1
direction: direction/reverse
top level: BBa_K1559003
start: 36
end: 47
orientation: inline

BBa_J23119
type: SequenceAnnotation
persistent identity: annotation2418638
display id: annotation2418638
version: 1
top level: BBa_K1559003
location: https://synbiohub.org/public/igem/BBa_K1559003/annotation2418638/range2418638/1
component: BBa_J23119

https://synbiohub.org/public/igem/BBa_K1559003/annotation2418638/range2418638/1
type: Range
persistent identity: range2418638
display id: range2418638
version: 1
direction: direction/reverse
top level: BBa_K1559003
start: 1
end: 35
orientation: inline

VP64
type: ComponentDefinition
persistent identity: BBa K1982012
display id: BBa_K1982012
version: 1
was generated by: https://synbiohub.org/public/igem/igem2sbol/1
description: VP64 transcription activitor
creator: Zexu Li
created: 2016-10-17T11:00:00Z
modified: 2016-10-18T09:58:31Z
discontinued: false
dominant: false
experience: experience/Works
group u list: _2449_
m user id: 30176
owner id: 30176
owning group id: 9
status: status/Planning
bookmark: false
mutable description: {| style="color:black" cellpadding="6" cellspacing="1" border="2" align="right" ! colspan="2" style="background:#FFBF00;"|VP64 |- |'''Function''' |transcription activator |- |'''RFC standard''' |[http://parts.igem.org/Help:Assembly_standard_10 RFC 10] |- |'''Backbone''' |pSB1C3 |- |'''Organism''' |''synthetic construct'' |- |'''Source''' |synthetic construct |- |'''Submitted by''' |[http://2016.igem.org/Team:NEU-China NEU-China 2016] |} Tetrameric VP16 transcription activator domain
mutable notes: synthetic construct
mutable provenance: synthetic construct
owned by: james
owned by: myers
star: false
type: DnaRegion
role: SO:0000417
role: partType/Protein Domain
sequence annotation: FLAG
sequence annotation: HA
sequence annotation: VP16
sequence annotation: Linker
sequence annotation: SV40 NLS
sequence annotation: TAATAA
sequence: https://synbiohub.org/public/igem/BBa_K1982012_sequence/1

FLAG
type: SequenceAnnotation
persistent identity: annotation2517156
display id: annotation2517156
version: 1
top level: VP64
role: SO:0000324
role: feature/tag
location: https://synbiohub.org/public/igem/BBa_K1982012/annotation2517156/range2517156/1

https://synbiohub.org/public/igem/BBa_K1982012/annotation2517156/range2517156/1
type: Range
persistent identity: range2517156
display id: range2517156
version: 1
direction: direction/forward
top level: VP64
start: 217
end: 240
orientation: inline

HA
type: SequenceAnnotation
persistent identity: annotation2517155
display id: annotation2517155
version: 1
top level: VP64
role: SO:0000324
role: feature/tag
location: https://synbiohub.org/public/igem/BBa_K1982012/annotation2517155/range2517155/1

https://synbiohub.org/public/igem/BBa_K1982012/annotation2517155/range2517155/1
type: Range
persistent identity: range2517155
display id: range2517155
version: 1
direction: direction/forward
top level: VP64
start: 187
end: 213
orientation: inline

VP16
type: SequenceAnnotation
persistent identity: annotation2517152
display id: annotation2517152
version: 1
top level: VP64
role: SO:0000110
role: feature/misc
location: https://synbiohub.org/public/igem/BBa_K1982012/annotation2517152/range2517152/1

https://synbiohub.org/public/igem/BBa_K1982012/annotation2517152/range2517152/1
type: Range
persistent identity: range2517152
display id: range2517152
version: 1
direction: direction/forward
top level: VP64
start: 103
end: 141
orientation: inline

Linker
type: SequenceAnnotation
persistent identity: annotation2517154
display id: annotation2517154
version: 1
top level: VP64
role: SO:0000110
role: feature/misc
location: https://synbiohub.org/public/igem/BBa_K1982012/annotation2517154/range2517154/1

https://synbiohub.org/public/igem/BBa_K1982012/annotation2517154/range2517154/1
type: Range
persistent identity: range2517154
display id: range2517154
version: 1
direction: direction/forward
top level: VP64
start: 181
end: 186
orientation: inline

SV40 NLS
type: SequenceAnnotation
persistent identity: annotation2517149
display id: annotation2517149
version: 1
top level: VP64
role: SO:0000110
role: feature/misc
location: https://synbiohub.org/public/igem/BBa_K1982012/annotation2517149/range2517149/1

https://synbiohub.org/public/igem/BBa_K1982012/annotation2517149/range2517149/1
type: Range
persistent identity: range2517149
display id: range2517149
version: 1
direction: direction/forward
top level: VP64
start: 1
end: 21
orientation: inline

TAATAA
type: SequenceAnnotation
persistent identity: annotation2517157
display id: annotation2517157
version: 1
top level: VP64
role: SO:0000319
role: feature/stop
location: https://synbiohub.org/public/igem/BBa_K1982012/annotation2517157/range2517157/1

https://synbiohub.org/public/igem/BBa_K1982012/annotation2517157/range2517157/1
type: Range
persistent identity: range2517157
display id: range2517157
version: 1
direction: direction/forward
top level: VP64
start: 241
end: 246
orientation: inline

VP16
type: SequenceAnnotation
persistent identity: annotation2517153
display id: annotation2517153
version: 1
top level: VP64
role: SO:0000110
role: feature/misc
location: https://synbiohub.org/public/igem/BBa_K1982012/annotation2517153/range2517153/1

https://synbiohub.org/public/igem/BBa_K1982012/annotation2517153/range2517153/1
type: Range
persistent identity: range2517153
display id: range2517153
version: 1
direction: direction/forward
top level: VP64
start: 142
end: 180
orientation: inline

VP16
type: SequenceAnnotation
persistent identity: annotation2517150
display id: annotation2517150
version: 1
top level: VP64
role: SO:0000110
role: feature/misc
location: https://synbiohub.org/public/igem/BBa_K1982012/annotation2517150/range2517150/1

https://synbiohub.org/public/igem/BBa_K1982012/annotation2517150/range2517150/1
type: Range
persistent identity: range2517150
display id: range2517150
version: 1
direction: direction/forward
top level: VP64
start: 22
end: 63
orientation: inline

VP16
type: SequenceAnnotation
persistent identity: annotation2517151
display id: annotation2517151
version: 1
top level: VP64
role: SO:0000110
role: feature/misc
location: https://synbiohub.org/public/igem/BBa_K1982012/annotation2517151/range2517151/1

https://synbiohub.org/public/igem/BBa_K1982012/annotation2517151/range2517151/1
type: Range
persistent identity: range2517151
display id: range2517151
version: 1
direction: direction/forward
top level: VP64
start: 64
end: 102
orientation: inline

BBa_K1150000
type: ComponentDefinition
persistent identity: BBa K1150000
display id: BBa_K1150000
version: 1
was generated by: https://synbiohub.org/public/igem/igem2sbol/1
description: dCas9
creator: Freiburg 2013
created: 2013-09-14T11:00:00Z
modified: 2015-05-08T01:09:27Z
discontinued: false
dominant: false
experience: experience/None
group u list: _1462_
m user id: 0
owner id: 16627
owning group id: 9
sample status: It's complicated
status: status/Planning
bookmark: false
mutable description: The human codon optimized Cas9 from the organism Streptococcus pyogenes was standardized (RFC 25). Additionally we mutated the Nickase function. In the end we engineered a DNA binding protein, that can be fused with different effectors in order to regulate gene expression.
mutable notes: Restriction sites were deleted by primer overhangs. Parts were fused together by fusion PCR. The nickase function was mutated in order to abbolish the demolition of DNA.
mutable provenance: Streptococcus pyogenes, Zhang lab
owned by: james
owned by: myers
star: false
type: DnaRegion
role: partType/Coding
role: SO:0000316
sequence annotation: Nickaseless mutation
sequence annotation: dCas9
sequence: https://synbiohub.org/public/igem/BBa_K1150000_sequence/1

Nickaseless mutation
type: SequenceAnnotation
persistent identity: annotation2354601
display id: annotation2354601
version: 1
top level: BBa_K1150000
role: SO:0001059
role: feature/mutation
location: https://synbiohub.org/public/igem/BBa_K1150000/annotation2354601/range2354601/1

https://synbiohub.org/public/igem/BBa_K1150000/annotation2354601/range2354601/1
type: Range
persistent identity: range2354601
display id: range2354601
version: 1
direction: direction/forward
top level: BBa_K1150000
start: 2515
end: 2517
orientation: inline

dCas9
type: SequenceAnnotation
persistent identity: annotation2347618
display id: annotation2347618
version: 1
top level: BBa_K1150000
role: feature/cds
role: SO:0000316
location: https://synbiohub.org/public/igem/BBa_K1150000/annotation2347618/range2347618/1

https://synbiohub.org/public/igem/BBa_K1150000/annotation2347618/range2347618/1
type: Range
persistent identity: range2347618
display id: range2347618
version: 1
direction: direction/forward
top level: BBa_K1150000
start: 1
end: 4101
orientation: inline

Cse1
type: ComponentDefinition
persistent identity: BBa K1218003
display id: BBa_K1218003
version: 1
was generated by: https://synbiohub.org/public/igem/igem2sbol/1
description: CRISPR CasA E. coli (Modern)
creator: Trevor Kalkus, Gordon Wade, Alissa Greenberg
created: 2013-08-27T11:00:00Z
modified: 2015-05-08T01:09:42Z
discontinued: false
dominant: false
experience: experience/None
group u list: _1532_
m user id: 0
owner id: 17712
owning group id: 9
sample status: In stock
status: status/Available
bookmark: false
mutable description: The E. coli CRISPR system uses the CASCADE complex to target invasive DNA. CasA is the first subunit of the CASCADE complex and is responsible for the initial identification of invasive DNA. This part can be used with casBCDE to create the full CASCADE protein complex.
mutable notes: We had to perform mutagenesis twice to remove PstI cut sites.
mutable provenance: E. coli DH5alpha genome
owned by: james
owned by: myers
star: false
type: DnaRegion
role: partType/Coding
role: SO:0000316
sequence: https://synbiohub.org/public/igem/BBa_K1218003_sequence/1

BBa_K1559007
type: ComponentDefinition
persistent identity: BBa K1559007
display id: BBa_K1559007
version: 1
was generated by: https://synbiohub.org/public/igem/igem2sbol/1
description: CRISPR/Cas9 system with pLac inducible promoter
creator: Xiuqi (Rex) Xia
created: 2014-10-12T11:00:00Z
modified: 2015-05-08T01:10:53Z
discontinued: false
dominant: false
group u list: _1944_
m user id: 0
owner id: 23047
owning group id: 9
sample status: Not in stock
status: status/Unavailable
bookmark: false
mutable description: TODO
mutable notes: TODO
mutable provenance: TODO
owned by: james
owned by: myers
star: false
type: DnaRegion
role: SO:0000804
role: partType/Generator
component: BBa_B0034
component: BBa_R0011
component: BBa_K1559002
sequence annotation: BBa_R0011
sequence annotation: BBa_B0034
sequence annotation: BBa_K1559002
sequence: https://synbiohub.org/public/igem/BBa_K1559007_sequence/1

BBa_B0034
type: Component
persistent identity: component2418672
display id: component2418672
version: 1
top level: BBa_K1559007
access: public
definition: https://synbiohub.org/public/igem/BBa_B0034/1

BBa_R0011
type: Component
persistent identity: component2418666
display id: component2418666
version: 1
top level: BBa_K1559007
access: public
definition: https://synbiohub.org/public/igem/BBa_R0011/1

BBa_K1559002
type: Component
persistent identity: component2418673
display id: component2418673
version: 1
top level: BBa_K1559007
access: public
definition: BBa_K1559002

BBa_R0011
type: SequenceAnnotation
persistent identity: annotation2418666
display id: annotation2418666
version: 1
top level: BBa_K1559007
location: https://synbiohub.org/public/igem/BBa_K1559007/annotation2418666/range2418666/1
component: BBa_R0011

https://synbiohub.org/public/igem/BBa_K1559007/annotation2418666/range2418666/1
type: Range
persistent identity: range2418666
display id: range2418666
version: 1
direction: direction/forward
top level: BBa_K1559007
start: 1
end: 54
orientation: inline

BBa_B0034
type: SequenceAnnotation
persistent identity: annotation2418672
display id: annotation2418672
version: 1
top level: BBa_K1559007
location: https://synbiohub.org/public/igem/BBa_K1559007/annotation2418672/range2418672/1
component: BBa_B0034

https://synbiohub.org/public/igem/BBa_K1559007/annotation2418672/range2418672/1
type: Range
persistent identity: range2418672
display id: range2418672
version: 1
direction: direction/reverse
top level: BBa_K1559007
start: 56
end: 67
orientation: inline

BBa_K1559002
type: SequenceAnnotation
persistent identity: annotation2418673
display id: annotation2418673
version: 1
top level: BBa_K1559007
location: https://synbiohub.org/public/igem/BBa_K1559007/annotation2418673/range2418673/1
component: BBa_K1559002

https://synbiohub.org/public/igem/BBa_K1559007/annotation2418673/range2418673/1
type: Range
persistent identity: range2418673
display id: range2418673
version: 1
direction: direction/reverse
top level: BBa_K1559007
start: 68
end: 5147
orientation: inline

BBa_K1129006
type: ComponentDefinition
persistent identity: BBa K1129006
display id: BBa_K1129006
version: 1
was generated by: https://synbiohub.org/public/igem/igem2sbol/1
description: Cas 9 from Streptococcus thermophilus
creator: UBC iGEM 2013
created: 2013-08-29T11:00:00Z
modified: 2015-05-08T01:09:19Z
discontinued: false
dominant: false
experience: experience/Works
group u list: _1441_
m user id: 0
owner id: 9774
owning group id: 9
sample status: It's complicated
status: status/Planning
bookmark: true
mutable description: Cas 9 from Streptococcus thermophilus
mutable notes: Illegal cut sites removed
mutable provenance: Streptococcus thermophilus
owned by: james
owned by: myers
star: false
type: DnaRegion
role: partType/Coding
role: SO:0000316
sequence annotation: Cas9
sequence: https://synbiohub.org/public/igem/BBa_K1129006_sequence/1

Cas9
type: SequenceAnnotation
persistent identity: annotation2430437
display id: annotation2430437
version: 1
top level: BBa_K1129006
role: feature/cds
role: SO:0000316
location: https://synbiohub.org/public/igem/BBa_K1129006/annotation2430437/range2430437/1

https://synbiohub.org/public/igem/BBa_K1129006/annotation2430437/range2430437/1
type: Range
persistent identity: range2430437
display id: range2430437
version: 1
direction: direction/forward
top level: BBa_K1129006
start: 1
end: 4167
orientation: inline

BBa_K1982010
type: ComponentDefinition
persistent identity: BBa K1982010
display id: BBa_K1982010
version: 1
was generated by: https://synbiohub.org/public/igem/igem2sbol/1
description: CRY2-VP64(Eukaryotic LACE system)
creator: Zexu Li
created: 2016-10-08T11:00:00Z
modified: 2016-10-18T10:53:31Z
discontinued: false
dominant: false
group u list: _2449_
m user id: 30176
owner id: 30176
owning group id: 9
status: status/Planning
bookmark: false
mutable 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''' |[http://parts.igem.org/Help:Assembly_standard_10 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 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. <!-- Add more about the biology of this part here ===Usage and Biology=== Protein data table for BioBrick <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_ ">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 MKMDKKTIVWFRRDL…
mutable notes: no
mutable provenance: CRY2 and CIB1 are extracted from Arabidopsis thaliana. tCas9 is from GE Share company. VP64 is synthetic construct.
owned by: james
owned by: myers
star: false
type: DnaRegion
role: partType/Coding
role: SO:0000316
sequence annotation: VP16
sequence annotation: CRY2
sequence annotation: ATG
sequence annotation: Eukaryotic rbs
sequence annotation: Linker
sequence annotation: TAA
sequence annotation: FLAG
sequence annotation: HA
sequence annotation: SV40 NLS
sequence: https://synbiohub.org/public/igem/BBa_K1982010_sequence/1

VP16
type: SequenceAnnotation
persistent identity: annotation2517286
display id: annotation2517286
version: 1
top level: BBa_K1982010
role: SO:0000110
role: feature/misc
location: https://synbiohub.org/public/igem/BBa_K1982010/annotation2517286/range2517286/1

https://synbiohub.org/public/igem/BBa_K1982010/annotation2517286/range2517286/1
type: Range
persistent identity: range2517286
display id: range2517286
version: 1
direction: direction/forward
top level: BBa_K1982010
start: 1954
end: 1992
orientation: inline

VP16
type: SequenceAnnotation
persistent identity: annotation2517284
display id: annotation2517284
version: 1
top level: BBa_K1982010
role: SO:0000110
role: feature/misc
location: https://synbiohub.org/public/igem/BBa_K1982010/annotation2517284/range2517284/1

https://synbiohub.org/public/igem/BBa_K1982010/annotation2517284/range2517284/1
type: Range
persistent identity: range2517284
display id: range2517284
version: 1
direction: direction/forward
top level: BBa_K1982010
start: 1876
end: 1914
orientation: inline

CRY2
type: SequenceAnnotation
persistent identity: annotation2517282
display id: annotation2517282
version: 1
top level: BBa_K1982010
role: SO:0000316
role: feature/protein
location: https://synbiohub.org/public/igem/BBa_K1982010/annotation2517282/range2517282/1

https://synbiohub.org/public/igem/BBa_K1982010/annotation2517282/range2517282/1
type: Range
persistent identity: range2517282
display id: range2517282
version: 1
direction: direction/forward
top level: BBa_K1982010
start: 22
end: 1854
orientation: inline

VP16
type: SequenceAnnotation
persistent identity: annotation2517285
display id: annotation2517285
version: 1
top level: BBa_K1982010
role: SO:0000110
role: feature/misc
location: https://synbiohub.org/public/igem/BBa_K1982010/annotation2517285/range2517285/1

https://synbiohub.org/public/igem/BBa_K1982010/annotation2517285/range2517285/1
type: Range
persistent identity: range2517285
display id: range2517285
version: 1
direction: direction/forward
top level: BBa_K1982010
start: 1915
end: 1953
orientation: inline

ATG
type: SequenceAnnotation
persistent identity: annotation2517281
display id: annotation2517281
version: 1
top level: BBa_K1982010
role: SO:0000318
role: feature/start
location: https://synbiohub.org/public/igem/BBa_K1982010/annotation2517281/range2517281/1

https://synbiohub.org/public/igem/BBa_K1982010/annotation2517281/range2517281/1
type: Range
persistent identity: range2517281
display id: range2517281
version: 1
direction: direction/forward
top level: BBa_K1982010
start: 19
end: 21
orientation: inline

Eukaryotic rbs
type: SequenceAnnotation
persistent identity: annotation2517280
display id: annotation2517280
version: 1
top level: BBa_K1982010
role: feature/rbs
role: SO:0000139
location: https://synbiohub.org/public/igem/BBa_K1982010/annotation2517280/range2517280/1

https://synbiohub.org/public/igem/BBa_K1982010/annotation2517280/range2517280/1
type: Range
persistent identity: range2517280
display id: range2517280
version: 1
direction: direction/forward
top level: BBa_K1982010
start: 1
end: 18
orientation: inline

Linker
type: SequenceAnnotation
persistent identity: annotation2517288
display id: annotation2517288
version: 1
top level: BBa_K1982010
role: SO:0000110
role: feature/misc
location: https://synbiohub.org/public/igem/BBa_K1982010/annotation2517288/range2517288/1

https://synbiohub.org/public/igem/BBa_K1982010/annotation2517288/range2517288/1
type: Range
persistent identity: range2517288
display id: range2517288
version: 1
direction: direction/forward
top level: BBa_K1982010
start: 2032
end: 2037
orientation: inline

VP16
type: SequenceAnnotation
persistent identity: annotation2517287
display id: annotation2517287
version: 1
top level: BBa_K1982010
role: SO:0000110
role: feature/misc
location: https://synbiohub.org/public/igem/BBa_K1982010/annotation2517287/range2517287/1

https://synbiohub.org/public/igem/BBa_K1982010/annotation2517287/range2517287/1
type: Range
persistent identity: range2517287
display id: range2517287
version: 1
direction: direction/forward
top level: BBa_K1982010
start: 1993
end: 2031
orientation: inline

TAA
type: SequenceAnnotation
persistent identity: annotation2517291
display id: annotation2517291
version: 1
top level: BBa_K1982010
role: SO:0000319
role: feature/stop
location: https://synbiohub.org/public/igem/BBa_K1982010/annotation2517291/range2517291/1

https://synbiohub.org/public/igem/BBa_K1982010/annotation2517291/range2517291/1
type: Range
persistent identity: range2517291
display id: range2517291
version: 1
direction: direction/forward
top level: BBa_K1982010
start: 2095
end: 2100
orientation: inline

FLAG
type: SequenceAnnotation
persistent identity: annotation2517290
display id: annotation2517290
version: 1
top level: BBa_K1982010
role: SO:0000324
role: feature/tag
location: https://synbiohub.org/public/igem/BBa_K1982010/annotation2517290/range2517290/1

https://synbiohub.org/public/igem/BBa_K1982010/annotation2517290/range2517290/1
type: Range
persistent identity: range2517290
display id: range2517290
version: 1
direction: direction/forward
top level: BBa_K1982010
start: 2071
end: 2094
orientation: inline

HA
type: SequenceAnnotation
persistent identity: annotation2517289
display id: annotation2517289
version: 1
top level: BBa_K1982010
role: SO:0000324
role: feature/tag
location: https://synbiohub.org/public/igem/BBa_K1982010/annotation2517289/range2517289/1

https://synbiohub.org/public/igem/BBa_K1982010/annotation2517289/range2517289/1
type: Range
persistent identity: range2517289
display id: range2517289
version: 1
direction: direction/forward
top level: BBa_K1982010
start: 2037
end: 2063
orientation: inline

SV40 NLS
type: SequenceAnnotation
persistent identity: annotation2517283
display id: annotation2517283
version: 1
top level: BBa_K1982010
role: SO:0000110
role: feature/misc
location: https://synbiohub.org/public/igem/BBa_K1982010/annotation2517283/range2517283/1

https://synbiohub.org/public/igem/BBa_K1982010/annotation2517283/range2517283/1
type: Range
persistent identity: range2517283
display id: range2517283
version: 1
direction: direction/forward
top level: BBa_K1982010
start: 1855
end: 1875
orientation: inline

BBa_K2017011
type: ComponentDefinition
persistent identity: BBa K2017011
display id: BBa_K2017011
version: 1
was generated by: https://synbiohub.org/public/igem/igem2sbol/1
description: 35s:5' + TFL consense + SAGTI-Luciferase + Tnos
creator: Monica Victoria Gutierrez Salazar
created: 2016-10-03T11:00:00Z
modified: 2016-10-22T05:15:34Z
discontinued: false
dominant: false
experience: experience/None
group u list: _2484_
m user id: 26626
owner id: 26626
owning group id: 9
status: status/Planning
bookmark: false
mutable description: Modul of the gRNA testing system created by Valencia UPV 2016 team. This system aims to test the functionality and efficiency of a chosen gRNA to target a particular gene. This system is modular, as the gene inserted can be chosen by the user, only taking into account the required overhangs (5'-AATG-3' and 5'-TTCG-3'). It includes a promoter 35s with a 5' region (BBa_K2017002), 20 nucleotides of the TFL consensus gene of Clemenules (clementine orange), the reporter luciferase with SAGTI linker and the terminator Tnos. Luciferase is out of its reading frame due to a nucleotide added upstream to the linker, and the initial ATG has been removed. This means that when the luciferase is translated, it will not be functional. To make it functional, it is necessary to make indels in the TFL fragment that put the luciferase in the correct reading frame. These indels can be made using the CRISPR/Cas9 system, which is the aim of this device.
mutable notes: It is important to notice that this device is modular. To insert the region of the gene of interest, this region must have the overhang 5'-AATG-3' and 5'-TTCG-3'.
mutable provenance: This part was obtained using GoldenBraid assembly. The Ga20ox consense sequence was obtained with oligonucleotide synthesis.
owned by: james
owned by: myers
star: false
type: DnaRegion
role: partType/Device
role: SO:0000804
component: BBa_K1484215
component: BBa_K2017003
component: BBa_K2017002
sequence annotation: BBa_K1484215
sequence annotation: BBa_K2017003
sequence annotation: BBa_K2017002
sequence: https://synbiohub.org/public/igem/BBa_K2017011_sequence/1

BBa_K1484215
type: Component
persistent identity: component2487442
display id: component2487442
version: 1
top level: BBa_K2017011
access: public
definition: https://synbiohub.org/public/igem/BBa_K1484215/1

BBa_K2017003
type: Component
persistent identity: component2487439
display id: component2487439
version: 1
top level: BBa_K2017011
access: public
definition: https://synbiohub.org/public/igem/BBa_K2017003/1

BBa_K2017002
type: Component
persistent identity: component2487436
display id: component2487436
version: 1
top level: BBa_K2017011
access: public
definition: https://synbiohub.org/public/igem/BBa_K2017002/1

BBa_K1484215
type: SequenceAnnotation
persistent identity: annotation2487442
display id: annotation2487442
version: 1
top level: BBa_K2017011
location: https://synbiohub.org/public/igem/BBa_K2017011/annotation2487442/range2487442/1
component: BBa_K1484215

https://synbiohub.org/public/igem/BBa_K2017011/annotation2487442/range2487442/1
type: Range
persistent identity: range2487442
display id: range2487442
version: 1
direction: direction/reverse
top level: BBa_K2017011
start: 2765
end: 3057
orientation: inline

BBa_K2017003
type: SequenceAnnotation
persistent identity: annotation2487439
display id: annotation2487439
version: 1
top level: BBa_K2017011
location: https://synbiohub.org/public/igem/BBa_K2017011/annotation2487439/range2487439/1
component: BBa_K2017003

https://synbiohub.org/public/igem/BBa_K2017011/annotation2487439/range2487439/1
type: Range
persistent identity: range2487439
display id: range2487439
version: 1
direction: direction/reverse
top level: BBa_K2017011
start: 1090
end: 2756
orientation: inline

BBa_K2017002
type: SequenceAnnotation
persistent identity: annotation2487436
display id: annotation2487436
version: 1
top level: BBa_K2017011
location: https://synbiohub.org/public/igem/BBa_K2017011/annotation2487436/range2487436/1
component: BBa_K2017002

https://synbiohub.org/public/igem/BBa_K2017011/annotation2487436/range2487436/1
type: Range
persistent identity: range2487436
display id: range2487436
version: 1
direction: direction/reverse
top level: BBa_K2017011
start: 1
end: 1081
orientation: inline

BBa_K1137013
type: ComponentDefinition
persistent identity: BBa K1137013
display id: BBa_K1137013
version: 1
was generated by: https://synbiohub.org/public/igem/igem2sbol/1
description: crRNA anti KAN
creator: Nicolas Koutsoubelis, Anne Loechner
created: 2013-10-03T11:00:00Z
modified: 2015-05-08T01:09:21Z
discontinued: false
dominant: false
experience: experience/None
group u list: _1449_
m user id: 0
owner id: 11926
owning group id: 9
sample status: It's complicated
status: status/Planning
bookmark: false
mutable description: This rather simple part contains the crRNA. The crRNA contains the target sequence to which the Cas9 protein is guided. The crRNA part can be cloned in front of any promoter. Note, that no RBS should be between the promoter and the crRNA. The crRNA is one of the two RNAs (crRNA and tracrRNA) that are needed to build the RNA dimer complex that fuses with the Cas9. The crRNA and the tracrRNA dimerize and are processed. This complex then binds to the CAS9. The RNA hybrid guides the Cas9 to its target and the Cas9 generates a site-specific double strand break. This crRNA here encodes for a sequence of the Kanamycin resistance gene cassette. The design of the crRNA is taken from the Jian et al. 2013 paper.
mutable notes: -
mutable provenance: This part was synthesized by IDT. THe design was taken from the paper below: Wenyan Jiang, David Bikard, David Cox, Feng Zhang & Luciano A Marraffini (2013): RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nature Biotechnology 31 (3), 233-239.
owned by: james
owned by: myers
star: false
type: DnaRegion
role: partType/Coding
role: SO:0000316
sequence: https://synbiohub.org/public/igem/BBa_K1137013_sequence/1

BBa_K1982006
type: ComponentDefinition
persistent identity: BBa K1982006
display id: BBa_K1982006
version: 1
was generated by: https://synbiohub.org/public/igem/igem2sbol/1
description: tCas9-Vp64(Prokaryotic)
creator: Zexu Li
created: 2016-09-11T11:00:00Z
modified: 2016-10-18T10:24:08Z
discontinued: false
dominant: false
group u list: _2449_
m user id: 30176
owner id: 30176
owning group id: 9
status: status/Planning
bookmark: false
mutable description: 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).
mutable notes: mutant EcoR 1 : TGTTGGGAATTc--->TGTTGGGAATTg
mutable provenance: tCas9 is from GE Share company. VP64 is synthetic construct.
owned by: james
owned by: myers
star: false
type: DnaRegion
role: partType/Device
role: SO:0000804
sequence annotation: VP16
sequence annotation: TAATAA
sequence annotation: ATG
sequence annotation: SV40 NLS
sequence annotation: Linker
sequence annotation: HA
sequence annotation: Prokaryotic rbs
sequence annotation: tCas9
sequence annotation: FLAG
sequence: https://synbiohub.org/public/igem/BBa_K1982006_sequence/1

VP16
type: SequenceAnnotation
persistent identity: annotation2517247
display id: annotation2517247
version: 1
top level: BBa_K1982006
role: SO:0000110
role: feature/misc
location: https://synbiohub.org/public/igem/BBa_K1982006/annotation2517247/range2517247/1

https://synbiohub.org/public/igem/BBa_K1982006/annotation2517247/range2517247/1
type: Range
persistent identity: range2517247
display id: range2517247
version: 1
direction: direction/forward
top level: BBa_K1982006
start: 4222
end: 4260
orientation: inline

VP16
type: SequenceAnnotation
persistent identity: annotation2517246
display id: annotation2517246
version: 1
top level: BBa_K1982006
role: SO:0000110
role: feature/misc
location: https://synbiohub.org/public/igem/BBa_K1982006/annotation2517246/range2517246/1

https://synbiohub.org/public/igem/BBa_K1982006/annotation2517246/range2517246/1
type: Range
persistent identity: range2517246
display id: range2517246
version: 1
direction: direction/forward
top level: BBa_K1982006
start: 4183
end: 4221
orientation: inline

TAATAA
type: SequenceAnnotation
persistent identity: annotation2517252
display id: annotation2517252
version: 1
top level: BBa_K1982006
role: SO:0000319
role: feature/stop
location: https://synbiohub.org/public/igem/BBa_K1982006/annotation2517252/range2517252/1

https://synbiohub.org/public/igem/BBa_K1982006/annotation2517252/range2517252/1
type: Range
persistent identity: range2517252
display id: range2517252
version: 1
direction: direction/forward
top level: BBa_K1982006
start: 4363
end: 4368
orientation: inline

VP16
type: SequenceAnnotation
persistent identity: annotation2517248
display id: annotation2517248
version: 1
top level: BBa_K1982006
role: SO:0000110
role: feature/misc
location: https://synbiohub.org/public/igem/BBa_K1982006/annotation2517248/range2517248/1

https://synbiohub.org/public/igem/BBa_K1982006/annotation2517248/range2517248/1
type: Range
persistent identity: range2517248
display id: range2517248
version: 1
direction: direction/forward
top level: BBa_K1982006
start: 4261
end: 4299
orientation: inline

ATG
type: SequenceAnnotation
persistent identity: annotation2517242
display id: annotation2517242
version: 1
top level: BBa_K1982006
role: SO:0000318
role: feature/start
location: https://synbiohub.org/public/igem/BBa_K1982006/annotation2517242/range2517242/1

https://synbiohub.org/public/igem/BBa_K1982006/annotation2517242/range2517242/1
type: Range
persistent identity: range2517242
display id: range2517242
version: 1
direction: direction/forward
top level: BBa_K1982006
start: 19
end: 21
orientation: inline

SV40 NLS
type: SequenceAnnotation
persistent identity: annotation2517244
display id: annotation2517244
version: 1
top level: BBa_K1982006
role: SO:0000110
role: feature/misc
location: https://synbiohub.org/public/igem/BBa_K1982006/annotation2517244/range2517244/1

https://synbiohub.org/public/igem/BBa_K1982006/annotation2517244/range2517244/1
type: Range
persistent identity: range2517244
display id: range2517244
version: 1
direction: direction/forward
top level: BBa_K1982006
start: 4123
end: 4143
orientation: inline

Linker
type: SequenceAnnotation
persistent identity: annotation2517249
display id: annotation2517249
version: 1
top level: BBa_K1982006
role: SO:0000110
role: feature/misc
location: https://synbiohub.org/public/igem/BBa_K1982006/annotation2517249/range2517249/1

https://synbiohub.org/public/igem/BBa_K1982006/annotation2517249/range2517249/1
type: Range
persistent identity: range2517249
display id: range2517249
version: 1
direction: direction/forward
top level: BBa_K1982006
start: 4300
end: 4305
orientation: inline

HA
type: SequenceAnnotation
persistent identity: annotation2517250
display id: annotation2517250
version: 1
top level: BBa_K1982006
role: SO:0000324
role: feature/tag
location: https://synbiohub.org/public/igem/BBa_K1982006/annotation2517250/range2517250/1

https://synbiohub.org/public/igem/BBa_K1982006/annotation2517250/range2517250/1
type: Range
persistent identity: range2517250
display id: range2517250
version: 1
direction: direction/forward
top level: BBa_K1982006
start: 4306
end: 4332
orientation: inline

VP16
type: SequenceAnnotation
persistent identity: annotation2517245
display id: annotation2517245
version: 1
top level: BBa_K1982006
role: SO:0000110
role: feature/misc
location: https://synbiohub.org/public/igem/BBa_K1982006/annotation2517245/range2517245/1

https://synbiohub.org/public/igem/BBa_K1982006/annotation2517245/range2517245/1
type: Range
persistent identity: range2517245
display id: range2517245
version: 1
direction: direction/forward
top level: BBa_K1982006
start: 4144
end: 4182
orientation: inline

Prokaryotic rbs
type: SequenceAnnotation
persistent identity: annotation2517241
display id: annotation2517241
version: 1
top level: BBa_K1982006
role: feature/rbs
role: SO:0000139
location: https://synbiohub.org/public/igem/BBa_K1982006/annotation2517241/range2517241/1

https://synbiohub.org/public/igem/BBa_K1982006/annotation2517241/range2517241/1
type: Range
persistent identity: range2517241
display id: range2517241
version: 1
direction: direction/forward
top level: BBa_K1982006
start: 1
end: 18
orientation: inline

tCas9
type: SequenceAnnotation
persistent identity: annotation2517243
display id: annotation2517243
version: 1
top level: BBa_K1982006
role: SO:0000316
role: feature/protein
location: https://synbiohub.org/public/igem/BBa_K1982006/annotation2517243/range2517243/1

https://synbiohub.org/public/igem/BBa_K1982006/annotation2517243/range2517243/1
type: Range
persistent identity: range2517243
display id: range2517243
version: 1
direction: direction/reverse
top level: BBa_K1982006
start: 22
end: 4122
orientation: inline

FLAG
type: SequenceAnnotation
persistent identity: annotation2517251
display id: annotation2517251
version: 1
top level: BBa_K1982006
role: SO:0000324
role: feature/tag
location: https://synbiohub.org/public/igem/BBa_K1982006/annotation2517251/range2517251/1

https://synbiohub.org/public/igem/BBa_K1982006/annotation2517251/range2517251/1
type: Range
persistent identity: range2517251
display id: range2517251
version: 1
direction: direction/forward
top level: BBa_K1982006
start: 4339
end: 4362
orientation: inline

BBa_K1081000
type: ComponentDefinition
persistent identity: BBa K1081000
display id: BBa_K1081000
version: 1
was generated by: https://synbiohub.org/public/igem/igem2sbol/1
description: J13002-dcas9
creator: Hangxing Jia
created: 2013-09-09T11:00:00Z
modified: 2015-05-08T01:09:04Z
discontinued: false
dominant: false
experience: experience/Works
group u list: _1390_
m user id: 0
owner id: 16101
owning group id: 9
sample status: It's complicated
status: status/Planning
bookmark: true
mutable description: Cas9 is an RNA-guided double-stranded DNA endonuclease, which belongs to the type II bacterial CRISPR system, from Streptococcus pyogenes. Dead cas9(dcas9), in which two mutations were introduced to abolish the endonuclease function, is added behind the biobrick J13002. Here we combine dcas9 with several different guide RNAs to activate or repress one of the double promoters??? transcription.
mutable notes: We made point mutation of EcoRI Restriction Enzyme cutting site inside the coding region of dcas9.
mutable provenance: Dcas9 was provided by addgene. Cas9 is a gene from Streptococcus pyogenes.
owned by: james
owned by: myers
star: false
type: DnaRegion
role: SO:0000804
role: partType/Composite
sequence annotation: dcas9
sequence annotation: BBa_K1081000
sequence annotation: B0034
sequence annotation: R0040
sequence annotation: BBa_J13002
sequence: https://synbiohub.org/public/igem/BBa_K1081000_sequence/1

dcas9
type: SequenceAnnotation
persistent identity: annotation2341843
display id: annotation2341843
version: 1
top level: BBa_K1081000
role: feature/cds
role: SO:0000316
location: https://synbiohub.org/public/igem/BBa_K1081000/annotation2341843/range2341843/1

https://synbiohub.org/public/igem/BBa_K1081000/annotation2341843/range2341843/1
type: Range
persistent identity: range2341843
display id: range2341843
version: 1
direction: direction/forward
top level: BBa_K1081000
start: 77
end: 4180
orientation: inline

BBa_K1081000
type: SequenceAnnotation
persistent identity: annotation2341841
display id: annotation2341841
version: 1
top level: BBa_K1081000
role: SO:0000804
role: feature/BioBrick
location: https://synbiohub.org/public/igem/BBa_K1081000/annotation2341841/range2341841/1

https://synbiohub.org/public/igem/BBa_K1081000/annotation2341841/range2341841/1
type: Range
persistent identity: range2341841
display id: range2341841
version: 1
direction: direction/reverse
top level: BBa_K1081000
start: 1
end: 4180
orientation: inline

B0034
type: SequenceAnnotation
persistent identity: annotation2341842
display id: annotation2341842
version: 1
top level: BBa_K1081000
role: SO:0000110
role: feature/misc
location: https://synbiohub.org/public/igem/BBa_K1081000/annotation2341842/range2341842/1

https://synbiohub.org/public/igem/BBa_K1081000/annotation2341842/range2341842/1
type: Range
persistent identity: range2341842
display id: range2341842
version: 1
direction: direction/forward
top level: BBa_K1081000
start: 63
end: 74
orientation: inline

R0040
type: SequenceAnnotation
persistent identity: annotation2341840
display id: annotation2341840
version: 1
top level: BBa_K1081000
role: SO:0000110
role: feature/misc
location: https://synbiohub.org/public/igem/BBa_K1081000/annotation2341840/range2341840/1

https://synbiohub.org/public/igem/BBa_K1081000/annotation2341840/range2341840/1
type: Range
persistent identity: range2341840
display id: range2341840
version: 1
direction: direction/forward
top level: BBa_K1081000
start: 1
end: 54
orientation: inline

BBa_J13002
type: SequenceAnnotation
persistent identity: annotation2341839
display id: annotation2341839
version: 1
top level: BBa_K1081000
role: SO:0000804
role: feature/BioBrick
location: https://synbiohub.org/public/igem/BBa_K1081000/annotation2341839/range2341839/1

https://synbiohub.org/public/igem/BBa_K1081000/annotation2341839/range2341839/1
type: Range
persistent identity: range2341839
display id: range2341839
version: 1
direction: direction/forward
top level: BBa_K1081000
start: 1
end: 74
orientation: inline

BBa_K1982005
type: ComponentDefinition
persistent identity: BBa K1982005
display id: BBa_K1982005
version: 1
was generated by: https://synbiohub.org/public/igem/igem2sbol/1
description: CRY2-VP64(Prokaryotic LACE system)
creator: Zexu Li
created: 2016-09-11T11:00:00Z
modified: 2016-10-18T10:38:55Z
discontinued: false
dominant: false
group u list: _2449_
m user id: 30176
owner id: 30176
owning group id: 9
status: status/Planning
bookmark: false
mutable description: 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.
mutable notes: no
mutable provenance: CRY2 and CIB1 are extracted from Arabidopsis thaliana. tCas9 is from GE Share company. VP64 is synthetic construct.
owned by: james
owned by: myers
star: false
type: DnaRegion
role: partType/Device
role: SO:0000804
sequence annotation: ATG
sequence annotation: VP16
sequence annotation: Linker
sequence annotation: HA
sequence annotation: FLAG
sequence annotation: SV40 NLS
sequence annotation: Prokaryotic rbs
sequence annotation: CRY2
sequence annotation: TAATAA
sequence: https://synbiohub.org/public/igem/BBa_K1982005_sequence/1

ATG
type: SequenceAnnotation
persistent identity: annotation2517263
display id: annotation2517263
version: 1
top level: BBa_K1982005
role: SO:0000318
role: feature/start
location: https://synbiohub.org/public/igem/BBa_K1982005/annotation2517263/range2517263/1

https://synbiohub.org/public/igem/BBa_K1982005/annotation2517263/range2517263/1
type: Range
persistent identity: range2517263
display id: range2517263
version: 1
direction: direction/forward
top level: BBa_K1982005
start: 19
end: 21
orientation: inline

VP16
type: SequenceAnnotation
persistent identity: annotation2517267
display id: annotation2517267
version: 1
top level: BBa_K1982005
role: SO:0000110
role: feature/misc
location: https://synbiohub.org/public/igem/BBa_K1982005/annotation2517267/range2517267/1

https://synbiohub.org/public/igem/BBa_K1982005/annotation2517267/range2517267/1
type: Range
persistent identity: range2517267
display id: range2517267
version: 1
direction: direction/forward
top level: BBa_K1982005
start: 1915
end: 1953
orientation: inline

VP16
type: SequenceAnnotation
persistent identity: annotation2517266
display id: annotation2517266
version: 1
top level: BBa_K1982005
role: SO:0000110
role: feature/misc
location: https://synbiohub.org/public/igem/BBa_K1982005/annotation2517266/range2517266/1

https://synbiohub.org/public/igem/BBa_K1982005/annotation2517266/range2517266/1
type: Range
persistent identity: range2517266
display id: range2517266
version: 1
direction: direction/forward
top level: BBa_K1982005
start: 1876
end: 1914
orientation: inline

Linker
type: SequenceAnnotation
persistent identity: annotation2517270
display id: annotation2517270
version: 1
top level: BBa_K1982005
role: SO:0000110
role: feature/misc
location: https://synbiohub.org/public/igem/BBa_K1982005/annotation2517270/range2517270/1

https://synbiohub.org/public/igem/BBa_K1982005/annotation2517270/range2517270/1
type: Range
persistent identity: range2517270
display id: range2517270
version: 1
direction: direction/forward
top level: BBa_K1982005
start: 2032
end: 2037
orientation: inline

HA
type: SequenceAnnotation
persistent identity: annotation2517271
display id: annotation2517271
version: 1
top level: BBa_K1982005
role: SO:0000324
role: feature/tag
location: https://synbiohub.org/public/igem/BBa_K1982005/annotation2517271/range2517271/1

https://synbiohub.org/public/igem/BBa_K1982005/annotation2517271/range2517271/1
type: Range
persistent identity: range2517271
display id: range2517271
version: 1
direction: direction/forward
top level: BBa_K1982005
start: 2037
end: 2063
orientation: inline

FLAG
type: SequenceAnnotation
persistent identity: annotation2517272
display id: annotation2517272
version: 1
top level: BBa_K1982005
role: SO:0000324
role: feature/tag
location: https://synbiohub.org/public/igem/BBa_K1982005/annotation2517272/range2517272/1

https://synbiohub.org/public/igem/BBa_K1982005/annotation2517272/range2517272/1
type: Range
persistent identity: range2517272
display id: range2517272
version: 1
direction: direction/forward
top level: BBa_K1982005
start: 2071
end: 2094
orientation: inline

SV40 NLS
type: SequenceAnnotation
persistent identity: annotation2517265
display id: annotation2517265
version: 1
top level: BBa_K1982005
role: SO:0000110
role: feature/misc
location: https://synbiohub.org/public/igem/BBa_K1982005/annotation2517265/range2517265/1

https://synbiohub.org/public/igem/BBa_K1982005/annotation2517265/range2517265/1
type: Range
persistent identity: range2517265
display id: range2517265
version: 1
direction: direction/forward
top level: BBa_K1982005
start: 1855
end: 1875
orientation: inline

Prokaryotic rbs
type: SequenceAnnotation
persistent identity: annotation2517262
display id: annotation2517262
version: 1
top level: BBa_K1982005
role: feature/rbs
role: SO:0000139
location: https://synbiohub.org/public/igem/BBa_K1982005/annotation2517262/range2517262/1

https://synbiohub.org/public/igem/BBa_K1982005/annotation2517262/range2517262/1
type: Range
persistent identity: range2517262
display id: range2517262
version: 1
direction: direction/forward
top level: BBa_K1982005
start: 1
end: 18
orientation: inline

VP16
type: SequenceAnnotation
persistent identity: annotation2517269
display id: annotation2517269
version: 1
top level: BBa_K1982005
role: SO:0000110
role: feature/misc
location: https://synbiohub.org/public/igem/BBa_K1982005/annotation2517269/range2517269/1

https://synbiohub.org/public/igem/BBa_K1982005/annotation2517269/range2517269/1
type: Range
persistent identity: range2517269
display id: range2517269
version: 1
direction: direction/forward
top level: BBa_K1982005
start: 1993
end: 2031
orientation: inline

CRY2
type: SequenceAnnotation
persistent identity: annotation2517264
display id: annotation2517264
version: 1
top level: BBa_K1982005
role: SO:0000316
role: feature/protein
location: https://synbiohub.org/public/igem/BBa_K1982005/annotation2517264/range2517264/1

https://synbiohub.org/public/igem/BBa_K1982005/annotation2517264/range2517264/1
type: Range
persistent identity: range2517264
display id: range2517264
version: 1
direction: direction/reverse
top level: BBa_K1982005
start: 22
end: 1854
orientation: inline

TAATAA
type: SequenceAnnotation
persistent identity: annotation2517273
display id: annotation2517273
version: 1
top level: BBa_K1982005
role: SO:0000319
role: feature/stop
location: https://synbiohub.org/public/igem/BBa_K1982005/annotation2517273/range2517273/1

https://synbiohub.org/public/igem/BBa_K1982005/annotation2517273/range2517273/1
type: Range
persistent identity: range2517273
display id: range2517273
version: 1
direction: direction/forward
top level: BBa_K1982005
start: 2095
end: 2100
orientation: inline

VP16
type: SequenceAnnotation
persistent identity: annotation2517268
display id: annotation2517268
version: 1
top level: BBa_K1982005
role: SO:0000110
role: feature/misc
location: https://synbiohub.org/public/igem/BBa_K1982005/annotation2517268/range2517268/1

https://synbiohub.org/public/igem/BBa_K1982005/annotation2517268/range2517268/1
type: Range
persistent identity: range2517268
display id: range2517268
version: 1
direction: direction/forward
top level: BBa_K1982005
start: 1954
end: 1992
orientation: inline

BBa_K2017009
type: ComponentDefinition
persistent identity: BBa K2017009
display id: BBa_K2017009
version: 1
was generated by: https://synbiohub.org/public/igem/igem2sbol/1
description: 35s + Ga20ox consense + AEK-Luciferase + Tnos
creator: Monica Victoria Gutierrez Salazar
created: 2016-10-03T11:00:00Z
modified: 2016-10-22T05:04:17Z
discontinued: false
dominant: false
group u list: _2484_
m user id: 26626
owner id: 26626
owning group id: 9
status: status/Planning
bookmark: false
mutable description: Modul of the gRNA testing system created by Valencia UPV 2016 team. This system aims to test the functionality and efficiency of a chosen gRNA to target a particular gene. This system is modular, as the gene inserted can be chosen by the user, only taking into account the required overhangs (5'-AATG-3' and 5'-TTCG-3'). It includes a promoter 35s (BBa_K1537015), 20 nucleotides of the Ga20ox consensus gene of Oryza sativa (rice), the reporter luciferase with AEK linker and the terminator Tnos. Luciferase is out of its reading frame due to a nucleotide added upstream to the linker, and the initial ATG has been removed. This means that when the luciferase is translated, it will not be functional. To make it functional, it is necessary to make indels in the Ga20ox fragment that put the luciferase in the correct reading frame. These indels can be made using the CRISPR/Cas9 system, which is the aim of this device.
mutable notes: This part was obtained using GoldenBraid assembly. The Ga20ox consense sequence was obtained with oligonucleotide synthesis.
mutable provenance: It is important to notice that this device is modular. To insert the region of the gene of interest, this region must have the overhang 5'-AATG-3' and 5'-TTCG-3'
owned by: james
owned by: myers
star: false
type: DnaRegion
role: partType/Device
role: SO:0000804
component: BBa_K1484215
component: BBa_K2017005
component: BBa_K1537015
sequence annotation: BBa_K1537015
sequence annotation: BBa_K2017005
sequence annotation: BBa_K1484215
sequence: https://synbiohub.org/public/igem/BBa_K2017009_sequence/1

BBa_K1484215
type: Component
persistent identity: component2487422
display id: component2487422
version: 1
top level: BBa_K2017009
access: public
definition: https://synbiohub.org/public/igem/BBa_K1484215/1

BBa_K2017005
type: Component
persistent identity: component2487419
display id: component2487419
version: 1
top level: BBa_K2017009
access: public
definition: https://synbiohub.org/public/igem/BBa_K2017005/1

BBa_K1537015
type: Component
persistent identity: component2487415
display id: component2487415
version: 1
top level: BBa_K2017009
access: public
definition: https://synbiohub.org/public/igem/BBa_K1537015/1

BBa_K1537015
type: SequenceAnnotation
persistent identity: annotation2487415
display id: annotation2487415
version: 1
top level: BBa_K2017009
location: https://synbiohub.org/public/igem/BBa_K2017009/annotation2487415/range2487415/1
component: BBa_K1537015

https://synbiohub.org/public/igem/BBa_K2017009/annotation2487415/range2487415/1
type: Range
persistent identity: range2487415
display id: range2487415
version: 1
direction: direction/reverse
top level: BBa_K2017009
start: 1
end: 860
orientation: inline

BBa_K2017005
type: SequenceAnnotation
persistent identity: annotation2487419
display id: annotation2487419
version: 1
top level: BBa_K2017009
location: https://synbiohub.org/public/igem/BBa_K2017009/annotation2487419/range2487419/1
component: BBa_K2017005

https://synbiohub.org/public/igem/BBa_K2017009/annotation2487419/range2487419/1
type: Range
persistent identity: range2487419
display id: range2487419
version: 1
direction: direction/reverse
top level: BBa_K2017009
start: 869
end: 2571
orientation: inline

BBa_K1484215
type: SequenceAnnotation
persistent identity: annotation2487422
display id: annotation2487422
version: 1
top level: BBa_K2017009
location: https://synbiohub.org/public/igem/BBa_K2017009/annotation2487422/range2487422/1
component: BBa_K1484215

https://synbiohub.org/public/igem/BBa_K2017009/annotation2487422/range2487422/1
type: Range
persistent identity: range2487422
display id: range2487422
version: 1
direction: direction/reverse
top level: BBa_K2017009
start: 2580
end: 2872
orientation: inline

BBa_K2017014
type: ComponentDefinition
persistent identity: BBa K2017014
display id: BBa_K2017014
version: 1
was generated by: https://synbiohub.org/public/igem/igem2sbol/1
description: 35s + TFL consense + RSIAT-TEV-Luciferase + Tnos
creator: Monica Victoria Gutierrez Salazar
created: 2016-10-03T11:00:00Z
modified: 2016-10-22T05:18:25Z
discontinued: false
dominant: false
group u list: _2484_
m user id: 26626
owner id: 26626
owning group id: 9
status: status/Planning
bookmark: false
mutable description: Modul of the gRNA testing system created by Valencia UPV 2016 team. This system aims to test the functionality and efficiency of a chosen gRNA to target a particular gene. This system is modular, as the gene inserted can be chosen by the user, only taking into account the required overhangs (5'-AATG-3' and 5'-TTCG-3'). It includes a promoter 35s (BBa_K1537015), 20 nucleotides of the TFL consensus gene of Clemenules (clementine orange), the reporter luciferase with RSIAT+TEV linker and the terminator Tnos. Luciferase is out of its reading frame due to a nucleotide added upstream to the linker, and the initial ATG has been removed. This means that when the luciferase is translated, it will not be functional. To make it functional, it is necessary to make indels in the TFL fragment that put the luciferase in the correct reading frame. These indels can be made using the CRISPR/Cas9 system, which is the aim of this device.
mutable notes: It is important to notice that this device is modular. To insert the region of the gene of interest, this region must have the overhang 5'-AATG-3' and 5'-TTCG-3'.
mutable provenance: This part was obtained using GoldenBraid assembly. The TFL consense sequence was obtained with oligonucleotide synthesis.
owned by: james
owned by: myers
star: false
type: DnaRegion
role: partType/Device
role: SO:0000804
component: BBa_K1537015
component: BBa_K1484215
component: BBa_K2017006
sequence annotation: BBa_K1484215
sequence annotation: BBa_K2017006
sequence annotation: BBa_K1537015
sequence: https://synbiohub.org/public/igem/BBa_K2017014_sequence/1

BBa_K1537015
type: Component
persistent identity: component2487468
display id: component2487468
version: 1
top level: BBa_K2017014
access: public
definition: https://synbiohub.org/public/igem/BBa_K1537015/1

BBa_K1484215
type: Component
persistent identity: component2487475
display id: component2487475
version: 1
top level: BBa_K2017014
access: public
definition: https://synbiohub.org/public/igem/BBa_K1484215/1

BBa_K2017006
type: Component
persistent identity: component2487472
display id: component2487472
version: 1
top level: BBa_K2017014
access: public
definition: https://synbiohub.org/public/igem/BBa_K2017006/1

BBa_K1484215
type: SequenceAnnotation
persistent identity: annotation2487475
display id: annotation2487475
version: 1
top level: BBa_K2017014
location: https://synbiohub.org/public/igem/BBa_K2017014/annotation2487475/range2487475/1
component: BBa_K1484215

https://synbiohub.org/public/igem/BBa_K2017014/annotation2487475/range2487475/1
type: Range
persistent identity: range2487475
display id: range2487475
version: 1
direction: direction/reverse
top level: BBa_K2017014
start: 2577
end: 2869
orientation: inline

BBa_K2017006
type: SequenceAnnotation
persistent identity: annotation2487472
display id: annotation2487472
version: 1
top level: BBa_K2017014
location: https://synbiohub.org/public/igem/BBa_K2017014/annotation2487472/range2487472/1
component: BBa_K2017006

https://synbiohub.org/public/igem/BBa_K2017014/annotation2487472/range2487472/1
type: Range
persistent identity: range2487472
display id: range2487472
version: 1
direction: direction/reverse
top level: BBa_K2017014
start: 869
end: 2568
orientation: inline

BBa_K1537015
type: SequenceAnnotation
persistent identity: annotation2487468
display id: annotation2487468
version: 1
top level: BBa_K2017014
location: https://synbiohub.org/public/igem/BBa_K2017014/annotation2487468/range2487468/1
component: BBa_K1537015

https://synbiohub.org/public/igem/BBa_K2017014/annotation2487468/range2487468/1
type: Range
persistent identity: range2487468
display id: range2487468
version: 1
direction: direction/reverse
top level: BBa_K2017014
start: 1
end: 860
orientation: inline

BBa_K1982009
type: ComponentDefinition
persistent identity: BBa K1982009
display id: BBa_K1982009
version: 1
was generated by: https://synbiohub.org/public/igem/igem2sbol/1
description: Eukaryotic Cryptochrome 2 (CRY2) ( a blue light stimulated photoreceptor)
creator: Zexu Li
created: 2016-10-08T11:00:00Z
modified: 2016-10-09T12:15:06Z
discontinued: false
dominant: false
group u list: _2449_
m user id: 30176
owner id: 30176
owning group id: 9
status: status/Planning
bookmark: false
mutable description: Cryptochrome 2 (CRY2) is a blue light stimulated photoreceptor, when exposed to blue light, it would interact withthe N-terminal fragment of CIB1 (CIBN) . This part is full-length CRY2 CDS. Optogenetic systems enable precise spatial and temporal control of cell behavior. A light-activated CRISPR/Cas9 effector (LACE) system that induces transcription of endogenous genes in the presence of blue light.This was accomplished by fusing the light-inducible heterodimerizing proteins CRY2 and CIB1 to a transactivation domain and the catalytically inactive tCas9, respectively. The versatile LACE system can be easily directed to new DNA sequences for the dynamic regulation of endogenous genes[1].
mutable notes: mutant EcoR 1 : TGTTGGGAATTc--->TGTTGGGAATTg
mutable provenance: The light-inducible heterodimerizering proteins CRY2 and CIB1 are both from Arabidopsis thaliana.
owned by: james
owned by: myers
star: false
type: DnaRegion
role: partType/Coding
role: SO:0000316
sequence annotation: Eukaryotic rbs
sequence annotation: atg
sequence: https://synbiohub.org/public/igem/BBa_K1982009_sequence/1

Eukaryotic rbs
type: SequenceAnnotation
persistent identity: annotation2488673
display id: annotation2488673
version: 1
top level: BBa_K1982009
role: feature/rbs
role: SO:0000139
location: https://synbiohub.org/public/igem/BBa_K1982009/annotation2488673/range2488673/1

https://synbiohub.org/public/igem/BBa_K1982009/annotation2488673/range2488673/1
type: Range
persistent identity: range2488673
display id: range2488673
version: 1
direction: direction/forward
top level: BBa_K1982009
start: 1
end: 18
orientation: inline

atg
type: SequenceAnnotation
persistent identity: annotation2488672
display id: annotation2488672
version: 1
top level: BBa_K1982009
role: SO:0000318
role: feature/start
location: https://synbiohub.org/public/igem/BBa_K1982009/annotation2488672/range2488672/1

https://synbiohub.org/public/igem/BBa_K1982009/annotation2488672/range2488672/1
type: Range
persistent identity: range2488672
display id: range2488672
version: 1
direction: direction/forward
top level: BBa_K1982009
start: 19
end: 21
orientation: inline

BBa_K2017001
type: ComponentDefinition
persistent identity: BBa K2017001
display id: BBa_K2017001
version: 1
was generated by: https://synbiohub.org/public/igem/igem2sbol/1
description: N-split Cas9 + DnaE N-intein
creator: Monica Victoria Gutierrez Salazar
created: 2016-10-02T11:00:00Z
modified: 2016-10-23T08:32:38Z
discontinued: false
dominant: false
experience: experience/Works
group u list: _2484_
m user id: 26626
owner id: 26626
owning group id: 9
status: status/Planning
bookmark: true
mutable description: Half of the SpCas9 protein fused to DnaE C-intein. The active full-lenght spCas9 can be recovered by using the other half of the protein, C-split Cas9 + DnaE C-intein. Inteins are used to fuse both parts of the protein, as they interact and splice, leaving the active protein.
mutable notes: As the part needs to be inserted in a viral vector, the overhangs have to be designed accordingly to the viral vector that is going to be used. The synthesis of N-split Cas9 could not be made in one gBlock, as it is too big. Hence, the half protein had to be splitted in two other parts for its synthesis.
mutable provenance: The protein domain is synthesized with the intein
owned by: james
owned by: myers
star: false
type: DnaRegion
role: SO:0000417
role: partType/Protein Domain
sequence annotation: N-split Cas9
sequence annotation: DnaE N-intein
sequence: https://synbiohub.org/public/igem/BBa_K2017001_sequence/1

N-split Cas9
type: SequenceAnnotation
persistent identity: annotation2485943
display id: annotation2485943
version: 1
top level: BBa_K2017001
role: SO:0000316
role: feature/protein
location: https://synbiohub.org/public/igem/BBa_K2017001/annotation2485943/range2485943/1

https://synbiohub.org/public/igem/BBa_K2017001/annotation2485943/range2485943/1
type: Range
persistent identity: range2485943
display id: range2485943
version: 1
direction: direction/forward
top level: BBa_K2017001
start: 1
end: 1933
orientation: inline

DnaE N-intein
type: SequenceAnnotation
persistent identity: annotation2485944
display id: annotation2485944
version: 1
top level: BBa_K2017001
role: SO:0000110
role: feature/misc
location: https://synbiohub.org/public/igem/BBa_K2017001/annotation2485944/range2485944/1

https://synbiohub.org/public/igem/BBa_K2017001/annotation2485944/range2485944/1
type: Range
persistent identity: range2485944
display id: range2485944
version: 1
direction: direction/forward
top level: BBa_K2017001
start: 1934
end: 2241
orientation: inline

Const gR4m
type: ComponentDefinition
persistent identity: BBa K1026002
display id: BBa_K1026002
version: 1
was generated by: https://synbiohub.org/public/igem/igem2sbol/1
description: Constitutively Expressed gRNA targeting mRFP
creator: Hongyi WU
created: 2013-09-15T11:00:00Z
modified: 2015-05-08T01:08:46Z
discontinued: false
dominant: false
experience: experience/Works
group u list: _1333_
m user id: 0
owner id: 12098
owning group id: 9
sample status: It's complicated
status: status/Planning
bookmark: true
mutable description: This Constitutively Expressed gRNA targeting mRFP is a no-BioBrick-scars assembly of the constitutive promoter BBa_J23100, DNA sequence of a gRNA that targets mRFP reporter and a reliable terminator BBa_B0015. This gRNA sequence comes from the following literature: QI, LEI S., LARSON, MATTHEW H., GILBERT, LUKE A., DOUDNA, JENNIFER A., WEISSMAN, JONATHAN S., ARKIN, ADAM P. & LIM, WENDELL A. 2013. Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression. Cell, 152, 1173-1183.
mutable notes: gRNA shall be exactly transcribed out. Any extra nucleotides would potentially jeopardize the normal function of the gRNA. Therefore, BioBrick scars are not allowed here. Instead of cut-and-ligation, inverse PCR or In-Fusion is preferred for the assembly task. In addition, a reliable terminator is applied here to ensure the termination of gRNA.
mutable provenance: The gRNA sequence is de novo synthesized.
owned by: james
owned by: myers
star: false
type: DnaRegion
role: SO:0000804
role: partType/Composite
component: BBa_J23100
component: BBa_K1026003
component: BBa_B0015
sequence annotation: BBa_K1026003
sequence annotation: BBa_J23100
sequence annotation: BBa_B0015
sequence: https://synbiohub.org/public/igem/BBa_K1026002_sequence/1

BBa_J23100
type: Component
persistent identity: component2347374
display id: component2347374
version: 1
top level: Const gR4m
access: public
definition: https://synbiohub.org/public/igem/BBa_J23100/1

BBa_K1026003
type: Component
persistent identity: component2347375
display id: component2347375
version: 1
top level: Const gR4m
access: public
definition: https://synbiohub.org/public/igem/BBa_K1026003/1

BBa_B0015
type: Component
persistent identity: component2347382
display id: component2347382
version: 1
top level: Const gR4m
access: public
definition: https://synbiohub.org/public/igem/BBa_B0015/1

BBa_K1026003
type: SequenceAnnotation
persistent identity: annotation2347375
display id: annotation2347375
version: 1
top level: Const gR4m
location: https://synbiohub.org/public/igem/BBa_K1026002/annotation2347375/range2347375/1
component: BBa_K1026003

https://synbiohub.org/public/igem/BBa_K1026002/annotation2347375/range2347375/1
type: Range
persistent identity: range2347375
display id: range2347375
version: 1
direction: direction/reverse
top level: Const gR4m
start: 36
end: 137
orientation: inline

BBa_J23100
type: SequenceAnnotation
persistent identity: annotation2347374
display id: annotation2347374
version: 1
top level: Const gR4m
location: https://synbiohub.org/public/igem/BBa_K1026002/annotation2347374/range2347374/1
component: BBa_J23100

https://synbiohub.org/public/igem/BBa_K1026002/annotation2347374/range2347374/1
type: Range
persistent identity: range2347374
display id: range2347374
version: 1
direction: direction/reverse
top level: Const gR4m
start: 1
end: 35
orientation: inline

BBa_B0015
type: SequenceAnnotation
persistent identity: annotation2347382
display id: annotation2347382
version: 1
top level: Const gR4m
location: https://synbiohub.org/public/igem/BBa_K1026002/annotation2347382/range2347382/1
component: BBa_B0015

https://synbiohub.org/public/igem/BBa_K1026002/annotation2347382/range2347382/1
type: Range
persistent identity: range2347382
display id: range2347382
version: 1
direction: direction/reverse
top level: Const gR4m
start: 138
end: 266
orientation: inline

BBa_K2017007
type: ComponentDefinition
persistent identity: BBa K2017007
display id: BBa_K2017007
version: 1
was generated by: https://synbiohub.org/public/igem/igem2sbol/1
description: 35s:5'+ Ga20ox consense + SAGTI-Luciferase + Tnos
creator: Monica Victoria Gutierrez Salazar
created: 2016-10-02T11:00:00Z
modified: 2016-10-22T04:50:07Z
discontinued: false
dominant: false
group u list: _2484_
m user id: 26626
owner id: 26626
owning group id: 9
status: status/Planning
bookmark: false
mutable description: Modul of the gRNA testing system created by Valencia UPV 2016 team. This system aims to test the functionality and efficiency of a chosen gRNA to target a particular gene. This system is modular, as the gene inserted can be chosen by the user, only taking into account the required overhangs (5'-AATG-3' and 5'-TTCG-3') It includes a promoter 35s with a 5' region (BBa_K2017002), 20 nucleotides of the Ga20ox consensus gene of Oryza sativa (rice), the reporter luciferase with SAGTI linker and the terminator Tnos. Luciferase is out of its reading frame due to a nucleotide added upstream to the linker, and the initial ATG has been removed. This means that when the luciferase is translated, it will not be functional. To make it functional, it is necessary to make indels in the Ga20ox fragment that put the luciferase in the correct reading frame. These indels can be made using the CRISPR/Cas9 system, which is the aim of this device.
mutable notes: It is important to notice that this device is modular. To insert the region of the gene of interest, this region must have the overhang 5'-AATG-3' and 5'-TTCG-3'
mutable provenance: This part was obtained using GoldenBraid assembly. The Ga20ox consense sequence was obtained with oligonucleotide synthesis.
owned by: james
owned by: myers
star: false
type: DnaRegion
role: partType/Device
role: SO:0000804
component: BBa_K1484215
component: BBa_K2017003
component: BBa_K2017002
sequence annotation: BBa_K2017002
sequence annotation: BBa_K1484215
sequence annotation: BBa_K2017003
sequence: https://synbiohub.org/public/igem/BBa_K2017007_sequence/1

BBa_K1484215
type: Component
persistent identity: component2487400
display id: component2487400
version: 1
top level: BBa_K2017007
access: public
definition: https://synbiohub.org/public/igem/BBa_K1484215/1

BBa_K2017003
type: Component
persistent identity: component2487397
display id: component2487397
version: 1
top level: BBa_K2017007
access: public
definition: https://synbiohub.org/public/igem/BBa_K2017003/1

BBa_K2017002
type: Component
persistent identity: component2487394
display id: component2487394
version: 1
top level: BBa_K2017007
access: public
definition: https://synbiohub.org/public/igem/BBa_K2017002/1

BBa_K2017002
type: SequenceAnnotation
persistent identity: annotation2487394
display id: annotation2487394
version: 1
top level: BBa_K2017007
location: https://synbiohub.org/public/igem/BBa_K2017007/annotation2487394/range2487394/1
component: BBa_K2017002

https://synbiohub.org/public/igem/BBa_K2017007/annotation2487394/range2487394/1
type: Range
persistent identity: range2487394
display id: range2487394
version: 1
direction: direction/reverse
top level: BBa_K2017007
start: 1
end: 1081
orientation: inline

BBa_K1484215
type: SequenceAnnotation
persistent identity: annotation2487400
display id: annotation2487400
version: 1
top level: BBa_K2017007
location: https://synbiohub.org/public/igem/BBa_K2017007/annotation2487400/range2487400/1
component: BBa_K1484215

https://synbiohub.org/public/igem/BBa_K2017007/annotation2487400/range2487400/1
type: Range
persistent identity: range2487400
display id: range2487400
version: 1
direction: direction/reverse
top level: BBa_K2017007
start: 2765
end: 3057
orientation: inline

BBa_K2017003
type: SequenceAnnotation
persistent identity: annotation2487397
display id: annotation2487397
version: 1
top level: BBa_K2017007
location: https://synbiohub.org/public/igem/BBa_K2017007/annotation2487397/range2487397/1
component: BBa_K2017003

https://synbiohub.org/public/igem/BBa_K2017007/annotation2487397/range2487397/1
type: Range
persistent identity: range2487397
display id: range2487397
version: 1
direction: direction/reverse
top level: BBa_K2017007
start: 1090
end: 2756
orientation: inline

BBa_K1994013
type: ComponentDefinition
persistent identity: BBa K1994013
display id: BBa_K1994013
version: 1
was generated by: https://synbiohub.org/public/igem/igem2sbol/1
description: sgRNA with dCas9 binding site sequence 9 and PP7 handle insert
creator: Liam Carroll
created: 2016-09-21T11:00:00Z
modified: 2016-10-19T09:23:58Z
discontinued: false
dominant: false
experience: experience/None
group u list: _2461_
m user id: 30870
owner id: 27126
owning group id: 9
status: status/Planning
bookmark: true
mutable description: For use in CRISPR/Cas9 systems. Contains golden gate adapter at 5' end for inserting target sequence. Also contains second golden gate adapter between truncated terminator and aspA terminator. Both golden gate adapters contain Bsa1 cut sites.
mutable notes: No special considerations
mutable provenance: Completely synthetic
owned by: james
owned by: myers
star: false
type: DnaRegion
role: SO:0000834
role: partType/RNA
sequence annotation: PP7 BP handle
sequence annotation: dCas9 handle
sequence annotation: truncated S. pyrogenes terminator
sequence annotation: 10bp Linker
sequence annotation: Sequence 9
sequence annotation: aspA terminator
sequence: https://synbiohub.org/public/igem/BBa_K1994013_sequence/1

PP7 BP handle
type: SequenceAnnotation
persistent identity: annotation2524244
display id: annotation2524244
version: 1
top level: BBa_K1994013
role: feature/stem loop
role: SO:0000313
location: https://synbiohub.org/public/igem/BBa_K1994013/annotation2524244/range2524244/1

https://synbiohub.org/public/igem/BBa_K1994013/annotation2524244/range2524244/1
type: Range
persistent identity: range2524244
display id: range2524244
version: 1
direction: direction/forward
top level: BBa_K1994013
start: 108
end: 138
orientation: inline

dCas9 handle
type: SequenceAnnotation
persistent identity: annotation2484494
display id: annotation2484494
version: 1
top level: BBa_K1994013
role: feature/stem loop
role: SO:0000313
location: https://synbiohub.org/public/igem/BBa_K1994013/annotation2484494/range2484494/1

https://synbiohub.org/public/igem/BBa_K1994013/annotation2484494/range2484494/1
type: Range
persistent identity: range2484494
display id: range2484494
version: 1
direction: direction/forward
top level: BBa_K1994013
start: 21
end: 62
orientation: inline

truncated S. pyrogenes terminator
type: SequenceAnnotation
persistent identity: annotation2484495
display id: annotation2484495
version: 1
top level: BBa_K1994013
role: feature/stem loop
role: SO:0000313
location: https://synbiohub.org/public/igem/BBa_K1994013/annotation2484495/range2484495/1

https://synbiohub.org/public/igem/BBa_K1994013/annotation2484495/range2484495/1
type: Range
persistent identity: range2484495
display id: range2484495
version: 1
direction: direction/forward
top level: BBa_K1994013
start: 63
end: 96
orientation: inline

10bp Linker
type: SequenceAnnotation
persistent identity: annotation2484496
display id: annotation2484496
version: 1
top level: BBa_K1994013
role: SO:0000110
role: feature/misc
location: https://synbiohub.org/public/igem/BBa_K1994013/annotation2484496/range2484496/1

https://synbiohub.org/public/igem/BBa_K1994013/annotation2484496/range2484496/1
type: Range
persistent identity: range2484496
display id: range2484496
version: 1
direction: direction/forward
top level: BBa_K1994013
start: 97
end: 107
orientation: inline

Sequence 9
type: SequenceAnnotation
persistent identity: annotation2484493
display id: annotation2484493
version: 1
top level: BBa_K1994013
role: SO:0000110
role: feature/misc
location: https://synbiohub.org/public/igem/BBa_K1994013/annotation2484493/range2484493/1

https://synbiohub.org/public/igem/BBa_K1994013/annotation2484493/range2484493/1
type: Range
persistent identity: range2484493
display id: range2484493
version: 1
direction: direction/forward
top level: BBa_K1994013
start: 1
end: 20
orientation: inline

aspA terminator
type: SequenceAnnotation
persistent identity: annotation2484497
display id: annotation2484497
version: 1
top level: BBa_K1994013
role: feature/stem loop
role: SO:0000313
location: https://synbiohub.org/public/igem/BBa_K1994013/annotation2484497/range2484497/1

https://synbiohub.org/public/igem/BBa_K1994013/annotation2484497/range2484497/1
type: Range
persistent identity: range2484497
display id: range2484497
version: 1
direction: direction/forward
top level: BBa_K1994013
start: 150
end: 189
orientation: inline

10bp Linker
type: SequenceAnnotation
persistent identity: annotation2524245
display id: annotation2524245
version: 1
top level: BBa_K1994013
role: SO:0000110
role: feature/misc
location: https://synbiohub.org/public/igem/BBa_K1994013/annotation2524245/range2524245/1

https://synbiohub.org/public/igem/BBa_K1994013/annotation2524245/range2524245/1
type: Range
persistent identity: range2524245
display id: range2524245
version: 1
direction: direction/forward
top level: BBa_K1994013
start: 139
end: 149
orientation: inline

BBa_K1994019
type: ComponentDefinition
persistent identity: BBa K1994019
display id: BBa_K1994019
version: 1
was generated by: https://synbiohub.org/public/igem/igem2sbol/1
description: Multiple dCas9 binding site sequence
creator: Liam Carroll
created: 2016-09-25T11:00:00Z
modified: 2016-10-19T03:50:49Z
discontinued: false
dominant: false
experience: experience/None
group u list: _2461_
m user id: 30870
owner id: 27126
owning group id: 9
status: status/Planning
bookmark: true
mutable description: Promoter sequence containing multiple PAMs staggered at regular intervals to provide multiple possible sites for dCas9 binding.
mutable notes: No special considerations
mutable provenance: Escherichia coli
owned by: james
owned by: myers
star: false
type: DnaRegion
role: SO:0000167
role: partType/Regulatory
sequence annotation: Multiple dCas9 binding sites
sequence annotation: 30nt dCas9 spacer
sequence: https://synbiohub.org/public/igem/BBa_K1994019_sequence/1

Multiple dCas9 binding sites
type: SequenceAnnotation
persistent identity: annotation2522793
display id: annotation2522793
version: 1
top level: BBa_K1994019
role: SO:0000110
role: feature/misc
location: https://synbiohub.org/public/igem/BBa_K1994019/annotation2522793/range2522793/1

https://synbiohub.org/public/igem/BBa_K1994019/annotation2522793/range2522793/1
type: Range
persistent identity: range2522793
display id: range2522793
version: 1
direction: direction/forward
top level: BBa_K1994019
start: 7
end: 210
orientation: inline

30nt dCas9 spacer
type: SequenceAnnotation
persistent identity: annotation2522794
display id: annotation2522794
version: 1
top level: BBa_K1994019
role: SO:0000110
role: feature/misc
location: https://synbiohub.org/public/igem/BBa_K1994019/annotation2522794/range2522794/1

https://synbiohub.org/public/igem/BBa_K1994019/annotation2522794/range2522794/1
type: Range
persistent identity: range2522794
display id: range2522794
version: 1
direction: direction/forward
top level: BBa_K1994019
start: 210
end: 239
orientation: inline

BBa_K2017008
type: ComponentDefinition
persistent identity: BBa K2017008
display id: BBa_K2017008
version: 1
was generated by: https://synbiohub.org/public/igem/igem2sbol/1
description: 35s + Ga20ox consense + RSIAT-Luciferase + Tnos
creator: Monica Victoria Gutierrez Salazar
created: 2016-10-03T11:00:00Z
modified: 2016-10-22T10:32:39Z
discontinued: false
dominant: false
experience: experience/None
group u list: _2484_
m user id: 26626
owner id: 26626
owning group id: 9
status: status/Planning
bookmark: false
mutable description: Modul of the gRNA testing system created by Valencia UPV 2016 team. This system aims to test the functionality and efficiency of a chosen gRNA to target a particular gene. This system is modular, as the gene inserted can be chosen by the user, only taking into account the required overhangs (5'-AATG-3' and 5'-TTCG-3'). It includes a promoter 35s with a 5' region (BBa_K2017002), 20 nucleotides of the Ga20ox consensus gene of Oryza sativa (rice), the reporter luciferase with RSIAT linker and the terminator Tnos. Luciferase is out of its reading frame due to a nucleotide added upstream to the linker, and the initial ATG has been removed. This means that when the luciferase is translated, it will not be functional. To make it functional, it is necessary to make indels in the Ga20ox fragment that put the luciferase in the correct reading frame. These indels can be made using the CRISPR/Cas9 system, which is the aim of this device.
mutable notes: It is important to notice that this device is modular. To insert the region of the gene of interest, this region must have the overhang 5'-AATG-3' and 5'-TTCG-3'
mutable provenance: This part was obtained using GoldenBraid assembly. The Ga20ox consense sequence was obtained with oligonucleotide synthesis.
owned by: james
owned by: myers
star: false
type: DnaRegion
role: partType/Device
role: SO:0000804
component: BBa_K1537015
component: BBa_K1484215
component: BBa_K2017004
sequence annotation: BBa_K1484215
sequence annotation: BBa_K1537015
sequence annotation: BBa_K2017004
sequence: https://synbiohub.org/public/igem/BBa_K2017008_sequence/1

BBa_K1537015
type: Component
persistent identity: component2487404
display id: component2487404
version: 1
top level: BBa_K2017008
access: public
definition: https://synbiohub.org/public/igem/BBa_K1537015/1

BBa_K1484215
type: Component
persistent identity: component2487411
display id: component2487411
version: 1
top level: BBa_K2017008
access: public
definition: https://synbiohub.org/public/igem/BBa_K1484215/1

BBa_K2017004
type: Component
persistent identity: component2487408
display id: component2487408
version: 1
top level: BBa_K2017008
access: public
definition: https://synbiohub.org/public/igem/BBa_K2017004/1

BBa_K1484215
type: SequenceAnnotation
persistent identity: annotation2487411
display id: annotation2487411
version: 1
top level: BBa_K2017008
location: https://synbiohub.org/public/igem/BBa_K2017008/annotation2487411/range2487411/1
component: BBa_K1484215

https://synbiohub.org/public/igem/BBa_K2017008/annotation2487411/range2487411/1
type: Range
persistent identity: range2487411
display id: range2487411
version: 1
direction: direction/reverse
top level: BBa_K2017008
start: 2544
end: 2836
orientation: inline

BBa_K1537015
type: SequenceAnnotation
persistent identity: annotation2487404
display id: annotation2487404
version: 1
top level: BBa_K2017008
location: https://synbiohub.org/public/igem/BBa_K2017008/annotation2487404/range2487404/1
component: BBa_K1537015

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type: Range
persistent identity: range2487404
display id: range2487404
version: 1
direction: direction/reverse
top level: BBa_K2017008
start: 1
end: 860
orientation: inline

BBa_K2017004
type: SequenceAnnotation
persistent identity: annotation2487408
display id: annotation2487408
version: 1
top level: BBa_K2017008
location: https://synbiohub.org/public/igem/BBa_K2017008/annotation2487408/range2487408/1
component: BBa_K2017004

https://synbiohub.org/public/igem/BBa_K2017008/annotation2487408/range2487408/1
type: Range
persistent identity: range2487408
display id: range2487408
version: 1
direction: direction/reverse
top level: BBa_K2017008
start: 869
end: 2535
orientation: inline

BBa_K1982008
type: ComponentDefinition
persistent identity: BBa K1982008
display id: BBa_K1982008
version: 1
was generated by: https://synbiohub.org/public/igem/igem2sbol/1
description: tCas9-CIBN (Eukaryotic LACE system)
creator: Zexu Li
created: 2016-10-08T11:00:00Z
modified: 2016-10-18T10:49:42Z
discontinued: false
dominant: false
group u list: _2449_
m user id: 30176
owner id: 30176
owning group id: 9
status: status/Planning
bookmark: false
mutable description: The NEU-China iGEM team 2016 designed a fusion protein consisting of tCas9 and CIBN for sequence-specific transactivation of a desired target locus (more information). We used our double truncated tCas9 (BBa_K1982007) impaired in its cleavage activity and fused it to the CIBN (BBa_K1982003). An prokaryotic RBS sequence from the Community collection(BBa_B0034) fused to the beginning of tCas9-CIBN. For detection of expression the fusion protein was tagged with a HA-epitope coding sequence (BBa_K1150016) .Figure 1 illustrates the detailed design of the whole device. Figure 1: Construct design. tCas9 was fused to CIBN. The resulting fusion construct was flanked by RBS sequences and tagged by a HA epitope. The pBad/araC promoter and rrnB T1 terminator were chosen to control gene expression. Optogenetic systems enable precise spatial and temporal control of cell behavior. A light-activated CRISPR/Cas9 effector (LACE) system that induces transcription of endogenous genes in the presence of blue light.This was accomplished by fusing the light-inducible heterodimerizing proteins CRY2 and CIB1 to a transactivation domain and the catalytically inactive tCas9, respectively. The versatile LACE system can be easily directed to new DNA sequences for the dynamic regulation of endogenous genes[1]. we fused the light-inducible heterodimerizering proteins CRY2 and CIB1 from Arabidopsis thaliana to the VP64 transactivation domain and C-terminus of tCas9.
mutable notes: 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.
mutable provenance: CRY2 and CIB1 are extracted from Arabidopsis thaliana. tCas9 is from GE Share company. VP64 is synthetic construct.
owned by: james
owned by: myers
star: false
type: DnaRegion
role: partType/Coding
role: SO:0000316
sequence annotation: TAATAA
sequence annotation: Eukaryotic rbs
sequence annotation: HA
sequence annotation: ATG
sequence annotation: BBa_K1982003
sequence annotation: tCas9
sequence: https://synbiohub.org/public/igem/BBa_K1982008_sequence/1

TAATAA
type: SequenceAnnotation
persistent identity: annotation2517278
display id: annotation2517278
version: 1
top level: BBa_K1982008
role: SO:0000319
role: feature/stop
location: https://synbiohub.org/public/igem/BBa_K1982008/annotation2517278/range2517278/1

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type: Range
persistent identity: range2517278
display id: range2517278
version: 1
direction: direction/forward
top level: BBa_K1982008
start: 4726
end: 4731
orientation: inline

Eukaryotic rbs
type: SequenceAnnotation
persistent identity: annotation2517274
display id: annotation2517274
version: 1
top level: BBa_K1982008
role: feature/rbs
role: SO:0000139
location: https://synbiohub.org/public/igem/BBa_K1982008/annotation2517274/range2517274/1

https://synbiohub.org/public/igem/BBa_K1982008/annotation2517274/range2517274/1
type: Range
persistent identity: range2517274
display id: range2517274
version: 1
direction: direction/forward
top level: BBa_K1982008
start: 1
end: 18
orientation: inline

HA
type: SequenceAnnotation
persistent identity: annotation2517279
display id: annotation2517279
version: 1
top level: BBa_K1982008
role: SO:0000324
role: feature/tag
location: https://synbiohub.org/public/igem/BBa_K1982008/annotation2517279/range2517279/1

https://synbiohub.org/public/igem/BBa_K1982008/annotation2517279/range2517279/1
type: Range
persistent identity: range2517279
display id: range2517279
version: 1
direction: direction/forward
top level: BBa_K1982008
start: 4669
end: 4725
orientation: inline

ATG
type: SequenceAnnotation
persistent identity: annotation2517276
display id: annotation2517276
version: 1
top level: BBa_K1982008
role: SO:0000318
role: feature/start
location: https://synbiohub.org/public/igem/BBa_K1982008/annotation2517276/range2517276/1

https://synbiohub.org/public/igem/BBa_K1982008/annotation2517276/range2517276/1
type: Range
persistent identity: range2517276
display id: range2517276
version: 1
direction: direction/forward
top level: BBa_K1982008
start: 19
end: 21
orientation: inline

BBa_K1982003
type: SequenceAnnotation
persistent identity: annotation2517277
display id: annotation2517277
version: 1
top level: BBa_K1982008
role: SO:0000804
role: feature/BioBrick
location: https://synbiohub.org/public/igem/BBa_K1982008/annotation2517277/range2517277/1

https://synbiohub.org/public/igem/BBa_K1982008/annotation2517277/range2517277/1
type: Range
persistent identity: range2517277
display id: range2517277
version: 1
direction: direction/forward
top level: BBa_K1982008
start: 4123
end: 4668
orientation: inline

tCas9
type: SequenceAnnotation
persistent identity: annotation2517275
display id: annotation2517275
version: 1
top level: BBa_K1982008
role: SO:0000316
role: feature/protein
location: https://synbiohub.org/public/igem/BBa_K1982008/annotation2517275/range2517275/1

https://synbiohub.org/public/igem/BBa_K1982008/annotation2517275/range2517275/1
type: Range
persistent identity: range2517275
display id: range2517275
version: 1
direction: direction/forward
top level: BBa_K1982008
start: 22
end: 4122
orientation: inline

BBa_K1150050
type: ComponentDefinition
persistent identity: BBa K1150050
display id: BBa_K1150050
version: 1
was generated by: https://synbiohub.org/public/igem/igem2sbol/1
description: Truncated CMV dCas9 Device #4
creator: Natalie Louis and Lisa Schmunk
created: 2013-09-16T11:00:00Z
modified: 2015-05-08T01:09:28Z
discontinued: false
dominant: false
experience: experience/None
group u list: _1462_
m user id: 0
owner id: 16627
owning group id: 9
sample status: It's complicated
status: status/Planning
bookmark: false
mutable description: .
mutable notes: .
mutable provenance: .
owned by: james
owned by: myers
star: false
type: DnaRegion
role: partType/Device
role: SO:0000804
component: BBa_K1150067
component: BBa_K747096
component: BBa_K1150010
component: BBa_K1150016
component: BBa_K1150012
sequence annotation: BBa_K747096
sequence annotation: BBa_K1150067
sequence annotation: BBa_K1150016
sequence annotation: BBa_K1150010
sequence annotation: BBa_K1150012
sequence: https://synbiohub.org/public/igem/BBa_K1150050_sequence/1

BBa_K1150067
type: Component
persistent identity: component2354585
display id: component2354585
version: 1
top level: BBa_K1150050
access: public
definition: https://synbiohub.org/public/igem/BBa_K1150067/1

BBa_K747096
type: Component
persistent identity: component2354580
display id: component2354580
version: 1
top level: BBa_K1150050
access: public
definition: https://synbiohub.org/public/igem/BBa_K747096/1

BBa_K1150010
type: Component
persistent identity: component2354587
display id: component2354587
version: 1
top level: BBa_K1150050
access: public
definition: https://synbiohub.org/public/igem/BBa_K1150010/1

BBa_K1150010
type: Component
persistent identity: component2354584
display id: component2354584
version: 1
top level: BBa_K1150050
access: public
definition: https://synbiohub.org/public/igem/BBa_K1150010/1

BBa_K1150016
type: Component
persistent identity: component2354582
display id: component2354582
version: 1
top level: BBa_K1150050
access: public
definition: https://synbiohub.org/public/igem/BBa_K1150016/1

BBa_K1150012
type: Component
persistent identity: component2354589
display id: component2354589
version: 1
top level: BBa_K1150050
access: public
definition: https://synbiohub.org/public/igem/BBa_K1150012/1

BBa_K747096
type: SequenceAnnotation
persistent identity: annotation2354580
display id: annotation2354580
version: 1
top level: BBa_K1150050
location: https://synbiohub.org/public/igem/BBa_K1150050/annotation2354580/range2354580/1
component: BBa_K747096

https://synbiohub.org/public/igem/BBa_K1150050/annotation2354580/range2354580/1
type: Range
persistent identity: range2354580
display id: range2354580
version: 1
direction: direction/reverse
top level: BBa_K1150050
start: 1
end: 580
orientation: inline

BBa_K1150067
type: SequenceAnnotation
persistent identity: annotation2354585
display id: annotation2354585
version: 1
top level: BBa_K1150050
location: https://synbiohub.org/public/igem/BBa_K1150050/annotation2354585/range2354585/1
component: BBa_K1150067

https://synbiohub.org/public/igem/BBa_K1150050/annotation2354585/range2354585/1
type: Range
persistent identity: range2354585
display id: range2354585
version: 1
direction: direction/reverse
top level: BBa_K1150050
start: 653
end: 3367
orientation: inline

BBa_K1150016
type: SequenceAnnotation
persistent identity: annotation2354582
display id: annotation2354582
version: 1
top level: BBa_K1150050
location: https://synbiohub.org/public/igem/BBa_K1150050/annotation2354582/range2354582/1
component: BBa_K1150016

https://synbiohub.org/public/igem/BBa_K1150050/annotation2354582/range2354582/1
type: Range
persistent identity: range2354582
display id: range2354582
version: 1
direction: direction/reverse
top level: BBa_K1150050
start: 589
end: 615
orientation: inline

BBa_K1150010
type: SequenceAnnotation
persistent identity: annotation2354587
display id: annotation2354587
version: 1
top level: BBa_K1150050
location: https://synbiohub.org/public/igem/BBa_K1150050/annotation2354587/range2354587/1
component: BBa_K1150010

https://synbiohub.org/public/igem/BBa_K1150050/annotation2354587/range2354587/1
type: Range
persistent identity: range2354587
display id: range2354587
version: 1
direction: direction/reverse
top level: BBa_K1150050
start: 3376
end: 3396
orientation: inline

BBa_K1150012
type: SequenceAnnotation
persistent identity: annotation2354589
display id: annotation2354589
version: 1
top level: BBa_K1150050
location: https://synbiohub.org/public/igem/BBa_K1150050/annotation2354589/range2354589/1
component: BBa_K1150012

https://synbiohub.org/public/igem/BBa_K1150050/annotation2354589/range2354589/1
type: Range
persistent identity: range2354589
display id: range2354589
version: 1
direction: direction/reverse
top level: BBa_K1150050
start: 3405
end: 3626
orientation: inline

BBa_K1150010
type: SequenceAnnotation
persistent identity: annotation2354584
display id: annotation2354584
version: 1
top level: BBa_K1150050
location: https://synbiohub.org/public/igem/BBa_K1150050/annotation2354584/range2354584/1
component: BBa_K1150010

https://synbiohub.org/public/igem/BBa_K1150050/annotation2354584/range2354584/1
type: Range
persistent identity: range2354584
display id: range2354584
version: 1
direction: direction/reverse
top level: BBa_K1150050
start: 624
end: 644
orientation: inline

BBa_K1150017
type: ComponentDefinition
persistent identity: BBa K1150017
display id: BBa_K1150017
version: 1
was generated by: https://synbiohub.org/public/igem/igem2sbol/1
description: dCas9 with CMV promoter
creator: Freiburg 2013
created: 2013-09-15T11:00:00Z
modified: 2015-05-08T01:09:27Z
discontinued: false
dominant: false
experience: experience/None
group u list: _1462_
m user id: 0
owner id: 16627
owning group id: 9
sample status: It's complicated
status: status/Planning
bookmark: false
mutable description: ..
mutable notes: ..
mutable provenance: ..
owned by: james
owned by: myers
star: false
type: DnaRegion
role: partType/Device
role: SO:0000804
component: BBa_K1150010
component: BBa_K747096
component: BBa_K1150016
component: BBa_K1150012
component: BBa_K1150000
sequence annotation: BBa_K1150010
sequence annotation: BBa_K1150012
sequence annotation: BBa_K1150016
sequence annotation: BBa_K747096
sequence annotation: BBa_K1150000
sequence: https://synbiohub.org/public/igem/BBa_K1150017_sequence/1

BBa_K1150010
type: Component
persistent identity: component2354367
display id: component2354367
version: 1
top level: BBa_K1150017
access: public
definition: https://synbiohub.org/public/igem/BBa_K1150010/1

BBa_K747096
type: Component
persistent identity: component2354363
display id: component2354363
version: 1
top level: BBa_K1150017
access: public
definition: https://synbiohub.org/public/igem/BBa_K747096/1

BBa_K1150016
type: Component
persistent identity: component2354365
display id: component2354365
version: 1
top level: BBa_K1150017
access: public
definition: https://synbiohub.org/public/igem/BBa_K1150016/1

BBa_K1150012
type: Component
persistent identity: component2354373
display id: component2354373
version: 1
top level: BBa_K1150017
access: public
definition: https://synbiohub.org/public/igem/BBa_K1150012/1

BBa_K1150000
type: Component
persistent identity: component2354369
display id: component2354369
version: 1
top level: BBa_K1150017
access: public
definition: BBa_K1150000

BBa_K1150010
type: Component
persistent identity: component2354371
display id: component2354371
version: 1
top level: BBa_K1150017
access: public
definition: https://synbiohub.org/public/igem/BBa_K1150010/1

BBa_K1150010
type: SequenceAnnotation
persistent identity: annotation2354371
display id: annotation2354371
version: 1
top level: BBa_K1150017
location: https://synbiohub.org/public/igem/BBa_K1150017/annotation2354371/range2354371/1
component: BBa_K1150010

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type: Range
persistent identity: range2354371
display id: range2354371
version: 1
direction: direction/reverse
top level: BBa_K1150017
start: 4762
end: 4782
orientation: inline

BBa_K1150012
type: SequenceAnnotation
persistent identity: annotation2354373
display id: annotation2354373
version: 1
top level: BBa_K1150017
location: https://synbiohub.org/public/igem/BBa_K1150017/annotation2354373/range2354373/1
component: BBa_K1150012

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type: Range
persistent identity: range2354373
display id: range2354373
version: 1
direction: direction/reverse
top level: BBa_K1150017
start: 4791
end: 5012
orientation: inline

BBa_K1150016
type: SequenceAnnotation
persistent identity: annotation2354365
display id: annotation2354365
version: 1
top level: BBa_K1150017
location: https://synbiohub.org/public/igem/BBa_K1150017/annotation2354365/range2354365/1
component: BBa_K1150016

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type: Range
persistent identity: range2354365
display id: range2354365
version: 1
direction: direction/reverse
top level: BBa_K1150017
start: 589
end: 615
orientation: inline

BBa_K1150010
type: SequenceAnnotation
persistent identity: annotation2354367
display id: annotation2354367
version: 1
top level: BBa_K1150017
location: https://synbiohub.org/public/igem/BBa_K1150017/annotation2354367/range2354367/1
component: BBa_K1150010

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type: Range
persistent identity: range2354367
display id: range2354367
version: 1
direction: direction/reverse
top level: BBa_K1150017
start: 624
end: 644
orientation: inline

BBa_K747096
type: SequenceAnnotation
persistent identity: annotation2354363
display id: annotation2354363
version: 1
top level: BBa_K1150017
location: https://synbiohub.org/public/igem/BBa_K1150017/annotation2354363/range2354363/1
component: BBa_K747096

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type: Range
persistent identity: range2354363
display id: range2354363
version: 1
direction: direction/reverse
top level: BBa_K1150017
start: 1
end: 580
orientation: inline

BBa_K1150000
type: SequenceAnnotation
persistent identity: annotation2354369
display id: annotation2354369
version: 1
top level: BBa_K1150017
location: https://synbiohub.org/public/igem/BBa_K1150017/annotation2354369/range2354369/1
component: BBa_K1150000

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type: Range
persistent identity: range2354369
display id: range2354369
version: 1
direction: direction/reverse
top level: BBa_K1150017
start: 653
end: 4753
orientation: inline

BBa_K1218011
type: ComponentDefinition
persistent identity: BBa K1218011
display id: BBa_K1218011
version: 1
was generated by: https://synbiohub.org/public/igem/igem2sbol/1
description: Cas9
creator: Sophia Liang
created: 2013-08-28T11:00:00Z
modified: 2015-05-08T01:09:42Z
discontinued: false
dominant: false
experience: experience/None
group u list: _1532_
m user id: 0
owner id: 17300
owning group id: 9
sample status: In stock
status: status/Available
bookmark: false
mutable description: CRISPR-Cas is a bacterial immune system that remembers and targets foreign viral DNA by storing DNA sequences, or spacers, between clustered regularly interspaced short palindromic repeats (CRISPRs). RNA transcripts of the spacers are then used to sense homologous DNA, which is cleaved by CRISPR-associated (Cas) proteins. This part codes for the tracrRNA, Cas9 protein, and minimal CRISPR array of a type II CRISPR-Cas system. The CRISPR array includes two CRISPR repeats separated by a spacer with two BsaI sites, which allow for the easy cloning of new spacers.
mutable notes: Mutagenesis to remove internal EcoRI site
mutable provenance: Streptococcus pyogenes
owned by: james
owned by: myers
star: false
type: DnaRegion
role: partType/Coding
role: SO:0000316
sequence: https://synbiohub.org/public/igem/BBa_K1218011_sequence/1

BBa_K2017012
type: ComponentDefinition
persistent identity: BBa K2017012
display id: BBa_K2017012
version: 1
was generated by: https://synbiohub.org/public/igem/igem2sbol/1
description: 35s + TFL consense + RSIAT-Luciferase + Tnos
creator: Monica Victoria Gutierrez Salazar
created: 2016-10-03T11:00:00Z
modified: 2016-10-22T05:15:51Z
discontinued: false
dominant: false
group u list: _2484_
m user id: 26626
owner id: 26626
owning group id: 9
status: status/Planning
bookmark: false
mutable description: Modul of the gRNA testing system created by Valencia UPV 2016 team. This system aims to test the functionality and efficiency of a chosen gRNA to target a particular gene. This system is modular, as the gene inserted can be chosen by the user, only taking into account the required overhangs (5'-AATG-3' and 5'-TTCG-3'). It includes a promoter 35s (BBa_K1537015), 20 nucleotides of the TFL consensus gene of Clemenules (clementine orange), the reporter luciferase with RSIAT linker and the terminator Tnos. Luciferase is out of its reading frame due to a nucleotide added upstream to the linker, and the initial ATG has been removed. This means that when the luciferase is translated, it will not be functional. To make it functional, it is necessary to make indels in the TFL fragment that put the luciferase in the correct reading frame. These indels can be made using the CRISPR/Cas9 system, which is the aim of this device.
mutable notes: It is important to notice that this device is modular. To insert the region of the gene of interest, this region must have the overhang 5'-AATG-3' and 5'-TTCG-3'.
mutable provenance: This part was obtained using GoldenBraid assembly. The TFL consense sequence was obtained with oligonucleotide synthesis.
owned by: james
owned by: myers
star: false
type: DnaRegion
role: partType/Device
role: SO:0000804
component: BBa_K2017004
component: BBa_K1537015
component: BBa_K1484215
sequence annotation: BBa_K1537015
sequence annotation: BBa_K1484215
sequence annotation: BBa_K2017004
sequence: https://synbiohub.org/public/igem/BBa_K2017012_sequence/1

BBa_K2017004
type: Component
persistent identity: component2487450
display id: component2487450
version: 1
top level: BBa_K2017012
access: public
definition: https://synbiohub.org/public/igem/BBa_K2017004/1

BBa_K1537015
type: Component
persistent identity: component2487446
display id: component2487446
version: 1
top level: BBa_K2017012
access: public
definition: https://synbiohub.org/public/igem/BBa_K1537015/1

BBa_K1484215
type: Component
persistent identity: component2487453
display id: component2487453
version: 1
top level: BBa_K2017012
access: public
definition: https://synbiohub.org/public/igem/BBa_K1484215/1

BBa_K1537015
type: SequenceAnnotation
persistent identity: annotation2487446
display id: annotation2487446
version: 1
top level: BBa_K2017012
location: https://synbiohub.org/public/igem/BBa_K2017012/annotation2487446/range2487446/1
component: BBa_K1537015

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type: Range
persistent identity: range2487446
display id: range2487446
version: 1
direction: direction/reverse
top level: BBa_K2017012
start: 1
end: 860
orientation: inline

BBa_K1484215
type: SequenceAnnotation
persistent identity: annotation2487453
display id: annotation2487453
version: 1
top level: BBa_K2017012
location: https://synbiohub.org/public/igem/BBa_K2017012/annotation2487453/range2487453/1
component: BBa_K1484215

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type: Range
persistent identity: range2487453
display id: range2487453
version: 1
direction: direction/reverse
top level: BBa_K2017012
start: 2544
end: 2836
orientation: inline

BBa_K2017004
type: SequenceAnnotation
persistent identity: annotation2487450
display id: annotation2487450
version: 1
top level: BBa_K2017012
location: https://synbiohub.org/public/igem/BBa_K2017012/annotation2487450/range2487450/1
component: BBa_K2017004

https://synbiohub.org/public/igem/BBa_K2017012/annotation2487450/range2487450/1
type: Range
persistent identity: range2487450
display id: range2487450
version: 1
direction: direction/reverse
top level: BBa_K2017012
start: 869
end: 2535
orientation: inline

BBa_K1559006
type: ComponentDefinition
persistent identity: BBa K1559006
display id: BBa_K1559006
version: 1
was generated by: https://synbiohub.org/public/igem/igem2sbol/1
description: CRISPR/Cas9 system with pBAD inducible promoter
creator: Xiuqi (Rex) Xia
created: 2014-10-12T11:00:00Z
modified: 2015-05-08T01:10:53Z
discontinued: false
dominant: false
group u list: _1944_
m user id: 0
owner id: 23047
owning group id: 9
sample status: Not in stock
status: status/Unavailable
bookmark: false
mutable description: TODO
mutable notes: TODO
mutable provenance: TODO
owned by: james
owned by: myers
star: false
type: DnaRegion
role: SO:0000804
role: partType/Generator
component: BBa_B0034
component: BBa_I13453
component: BBa_K1559002
sequence annotation: BBa_K1559002
sequence annotation: BBa_I13453
sequence annotation: BBa_B0034
sequence: https://synbiohub.org/public/igem/BBa_K1559006_sequence/1

BBa_B0034
type: Component
persistent identity: component2418656
display id: component2418656
version: 1
top level: BBa_K1559006
access: public
definition: https://synbiohub.org/public/igem/BBa_B0034/1

BBa_I13453
type: Component
persistent identity: component2418654
display id: component2418654
version: 1
top level: BBa_K1559006
access: public
definition: https://synbiohub.org/public/igem/BBa_I13453/1

BBa_K1559002
type: Component
persistent identity: component2418657
display id: component2418657
version: 1
top level: BBa_K1559006
access: public
definition: BBa_K1559002

BBa_K1559002
type: SequenceAnnotation
persistent identity: annotation2418657
display id: annotation2418657
version: 1
top level: BBa_K1559006
location: https://synbiohub.org/public/igem/BBa_K1559006/annotation2418657/range2418657/1
component: BBa_K1559002

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type: Range
persistent identity: range2418657
display id: range2418657
version: 1
direction: direction/reverse
top level: BBa_K1559006
start: 143
end: 5222
orientation: inline

BBa_I13453
type: SequenceAnnotation
persistent identity: annotation2418654
display id: annotation2418654
version: 1
top level: BBa_K1559006
location: https://synbiohub.org/public/igem/BBa_K1559006/annotation2418654/range2418654/1
component: BBa_I13453

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type: Range
persistent identity: range2418654
display id: range2418654
version: 1
direction: direction/reverse
top level: BBa_K1559006
start: 1
end: 130
orientation: inline

BBa_B0034
type: SequenceAnnotation
persistent identity: annotation2418656
display id: annotation2418656
version: 1
top level: BBa_K1559006
location: https://synbiohub.org/public/igem/BBa_K1559006/annotation2418656/range2418656/1
component: BBa_B0034

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type: Range
persistent identity: range2418656
display id: range2418656
version: 1
direction: direction/reverse
top level: BBa_K1559006
start: 131
end: 142
orientation: inline

BBa_K1982007
type: ComponentDefinition
persistent identity: BBa K1982007
display id: BBa_K1982007
version: 1
was generated by: https://synbiohub.org/public/igem/igem2sbol/1
description: Eukaryotic tCAS9
creator: Zexu Li
created: 2016-10-07T11:00:00Z
modified: 2016-10-09T12:04:45Z
discontinued: false
dominant: false
group u list: _2449_
m user id: 30176
owner id: 30176
owning group id: 9
status: status/Planning
bookmark: false
mutable description: tCas9 is a standardized (RFC 10) protein.Interacting with a DNA-binding RNA and fused with different effector domains it can be used for specific gene regulation. Cas9 is the main protein of the CRISPR/Cas system II of Streptococcus pyogenes. CRISPR systems protect bacteria and archaea from phages by recognizing and cleaving of invading phage DNA.This recognition is based on Watson Crick base pairing between a short RNA, called crRNA, and the complementary DNA strand. A second RNA, called tracrRNA, connects crRNA and Cas9. These three parts together form a protein-RNA-DNA complex with the targeted DNA strand [1] Cas9 became of great interest for research concerning DNA targeting, because of its ability to recognize site specific DNA strands by a crRNA. At first the functionality of Cas9 was modified by exchanging aminoacids. As a result, Cas9 was able to introduce mutations within the genome of several organisms by causing double strand breaks [2][3]. Then, it was converted from a nuclease to a nickase introducing single strand breaks [4] and lately it was converted to an enzymatically inactive form, called dCas9 [5]. This tCas9 is standardized (RFC 10). It can be used as a DNA binding protein, that can be fused with different effectors in order to regulate gene expression.
mutable notes: mutant Pst 1 :gctacctgcaG to gctacctgcaC mutant Pst 1 :actatctgcaG to actatctgcaC
mutable provenance: plasmid from GE Share company. [1] Westra E.R., Swarts D.C., Staals R.H., Jore M.M., Brouns S.J., van der Oost J. (2012). The CRISPRs, they are a-changin': how prokaryotes generate adaptive immunity. Annu Rev Genet. 46, 311-39 [2] Mali P., Yang L., Esvelt K.M., Aach J., Guell M., DiCarlo J.E., Norville J.E., Church G.M. (2013). RNA-guided human genome engineering via Cas9. Science 339(6121), 823-6 [3] Jiang W., Bikard D., Cox D., Zhang F., Marraffini L.A. (2013). RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol. 31(3), 233-9 [4] Cong, L., Ran, F.A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P.D., Wu, X., Jiang, W., Marraffini, L.A., Zhang, F. (2013). Multiplex Genome Engineering Using CRISPR/Cas Systems. Science 339 (6121), 819-23 [5] Qi L.S., Larson M.H., Gilbert L.A., Doudna J.A., Weissman J.S., Arkin A.P., Lim W.A. (2013). Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152(5), 1173-83
owned by: james
owned by: myers
star: false
type: DnaRegion
role: partType/Coding
role: SO:0000316
sequence annotation: Eukaryotic rbs
sequence annotation: atg
sequence: https://synbiohub.org/public/igem/BBa_K1982007_sequence/1

Eukaryotic rbs
type: SequenceAnnotation
persistent identity: annotation2488563
display id: annotation2488563
version: 1
top level: BBa_K1982007
role: feature/rbs
role: SO:0000139
location: https://synbiohub.org/public/igem/BBa_K1982007/annotation2488563/range2488563/1

https://synbiohub.org/public/igem/BBa_K1982007/annotation2488563/range2488563/1
type: Range
persistent identity: range2488563
display id: range2488563
version: 1
direction: direction/forward
top level: BBa_K1982007
start: 1
end: 18
orientation: inline

atg
type: SequenceAnnotation
persistent identity: annotation2488564
display id: annotation2488564
version: 1
top level: BBa_K1982007
role: SO:0000318
role: feature/start
location: https://synbiohub.org/public/igem/BBa_K1982007/annotation2488564/range2488564/1

https://synbiohub.org/public/igem/BBa_K1982007/annotation2488564/range2488564/1
type: Range
persistent identity: range2488564
display id: range2488564
version: 1
direction: direction/forward
top level: BBa_K1982007
start: 19
end: 21
orientation: inline

Prokaryoti
type: ComponentDefinition
persistent identity: BBa K1982001
display id: BBa_K1982001
version: 1
was generated by: https://synbiohub.org/public/igem/igem2sbol/1
description: Prokaryotic tCAS9
creator: Zexu Li
created: 2016-09-09T11:00:00Z
modified: 2016-10-09T12:35:07Z
discontinued: false
dominant: false
experience: experience/None
group u list: _2449_
m user id: 30176
owner id: 30176
owning group id: 9
status: status/Planning
bookmark: false
mutable description: tCas9 is a standardized (RFC 10) protein.Interacting with a DNA-binding RNA and fused with different effector domains it can be used for specific gene regulation. Cas9 is the main protein of the CRISPR/Cas system II of Streptococcus pyogenes. CRISPR systems protect bacteria and archaea from phages by recognizing and cleaving of invading phage DNA.This recognition is based on Watson Crick base pairing between a short RNA, called crRNA, and the complementary DNA strand. A second RNA, called tracrRNA, connects crRNA and Cas9. These three parts together form a protein-RNA-DNA complex with the targeted DNA strand [1] Cas9 became of great interest for research concerning DNA targeting, because of its ability to recognize site specific DNA strands by a crRNA. At first the functionality of Cas9 was modified by exchanging aminoacids. As a result, Cas9 was able to introduce mutations within the genome of several organisms by causing double strand breaks [2][3]. Then, it was converted from a nuclease to a nickase introducing single strand breaks [4] and lately it was converted to an enzymatically inactive form, called dCas9 [5]. This tCas9 is standardized (RFC 10). It can be used as a DNA binding protein, that can be fused with different effectors in order to regulate gene expression.
mutable notes: mutant Pst 1 :gctacctgcaG to gctacctgcaC mutant Pst 1 :actatctgcaG to actatctgcaC
mutable provenance: GE Share [1] Westra E.R., Swarts D.C., Staals R.H., Jore M.M., Brouns S.J., van der Oost J. (2012). The CRISPRs, they are a-changin': how prokaryotes generate adaptive immunity. Annu Rev Genet. 46, 311-39 [2] Mali P., Yang L., Esvelt K.M., Aach J., Guell M., DiCarlo J.E., Norville J.E., Church G.M. (2013). RNA-guided human genome engineering via Cas9. Science 339(6121), 823-6 [3] Jiang W., Bikard D., Cox D., Zhang F., Marraffini L.A. (2013). RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol. 31(3), 233-9 [4] Cong, L., Ran, F.A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P.D., Wu, X., Jiang, W., Marraffini, L.A., Zhang, F. (2013). Multiplex Genome Engineering Using CRISPR/Cas Systems. Science 339 (6121), 819-23 [5] Qi L.S., Larson M.H., Gilbert L.A., Doudna J.A., Weissman J.S., Arkin A.P., Lim W.A. (2013). Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152(5), 1173-83
owned by: james
owned by: myers
star: false
type: DnaRegion
role: partType/Coding
role: SO:0000316
sequence annotation: ATG
sequence annotation: Prokaryotic rbs
sequence: https://synbiohub.org/public/igem/BBa_K1982001_sequence/1

ATG
type: SequenceAnnotation
persistent identity: annotation2482946
display id: annotation2482946
version: 1
top level: Prokaryoti
role: SO:0000318
role: feature/start
location: https://synbiohub.org/public/igem/BBa_K1982001/annotation2482946/range2482946/1

https://synbiohub.org/public/igem/BBa_K1982001/annotation2482946/range2482946/1
type: Range
persistent identity: range2482946
display id: range2482946
version: 1
direction: direction/forward
top level: Prokaryoti
start: 18
end: 21
orientation: inline

Prokaryotic rbs
type: SequenceAnnotation
persistent identity: annotation2488562
display id: annotation2488562
version: 1
top level: Prokaryoti
role: feature/rbs
role: SO:0000139
location: https://synbiohub.org/public/igem/BBa_K1982001/annotation2488562/range2488562/1

https://synbiohub.org/public/igem/BBa_K1982001/annotation2488562/range2488562/1
type: Range
persistent identity: range2488562
display id: range2488562
version: 1
direction: direction/forward
top level: Prokaryoti
start: 1
end: 18
orientation: inline

BBa_K1160000
type: ComponentDefinition
persistent identity: BBa K1160000
display id: BBa_K1160000
version: 1
was generated by: https://synbiohub.org/public/igem/igem2sbol/1
description: coding sequence of Cas9 from CRISPR system type II
creator: Huang Xingxu
created: 2013-09-12T11:00:00Z
modified: 2015-05-08T01:09:32Z
discontinued: false
dominant: false
experience: experience/None
group u list: _1472_
m user id: 0
owner id: 16689
owning group id: 9
sample status: It's complicated
status: status/Planning
bookmark: false
mutable description: CRISPR (clustered regularly interspaced short palindromic repeat) is an adaptive immune system provide protection against mobile genetic element by guided specific DNA cleavage. Cas9 (CRISPR-associated protein 9) belongs to type II CRISPR system and contains HNH motif and three RuvC-like motifs, homologous to HNH and RuvC endonuclease, respectively. Cas9 has strong DNA cleavage activity, thus it showed potential use in gene editing. This part contains the coding sequence of cas9 from Streptococcus pyogenes (UniProtKB AC number:Q99ZW2). Some "redundant" sequence was added to ensure the Cas9's normal function or just for the convenience to detect it in cell. The cas9 protein must be guided by two RNAs, crRNA(CRISPR RNA) and tracrRNA(trans-activating crRNA), to complete its specific DNA cleavage function. We build another part-a chimeic RNA-that combines the feature two RNAs. For more details, refer to part BBa_K1160001. With this part, you can 1)directly use it to express cas9 protein in cell or 2)use T7 RNA polymerase to get cas9's mRNA, which can be further modified to prepare authentic mRNA that can be used for translation in vivo.
mutable notes: to be added
mutable provenance: Streptococcus pyogenes
owned by: james
owned by: myers
star: false
type: DnaRegion
role: SO:0000155
role: partType/Plasmid
sequence annotation: AmpR
sequence annotation: Ca9 coding sequence
sequence annotation: f1 ori
sequence annotation: ori
sequence annotation: CMV enhancer
sequence annotation: BSD
sequence annotation: bGH poly(A) signal
sequence annotation: CMV promoter
sequence: https://synbiohub.org/public/igem/BBa_K1160000_sequence/1

AmpR
type: SequenceAnnotation
persistent identity: annotation2363688
display id: annotation2363688
version: 1
top level: BBa_K1160000
role: SO:0000316
role: feature/protein
location: https://synbiohub.org/public/igem/BBa_K1160000/annotation2363688/range2363688/1

https://synbiohub.org/public/igem/BBa_K1160000/annotation2363688/range2363688/1
type: Range
persistent identity: range2363688
display id: range2363688
version: 1
direction: direction/forward
top level: BBa_K1160000
start: 8163
end: 9023
orientation: inline

Ca9 coding sequence
type: SequenceAnnotation
persistent identity: annotation2363681
display id: annotation2363681
version: 1
top level: BBa_K1160000
role: SO:0000316
role: feature/protein
location: https://synbiohub.org/public/igem/BBa_K1160000/annotation2363681/range2363681/1

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type: Range
persistent identity: range2363681
display id: range2363681
version: 1
direction: direction/forward
top level: BBa_K1160000
start: 904
end: 5010
orientation: inline

f1 ori
type: SequenceAnnotation
persistent identity: annotation2363685
display id: annotation2363685
version: 1
top level: BBa_K1160000
role: SO:0000110
role: feature/misc
location: https://synbiohub.org/public/igem/BBa_K1160000/annotation2363685/range2363685/1

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type: Range
persistent identity: range2363685
display id: range2363685
version: 1
direction: direction/forward
top level: BBa_K1160000
start: 5390
end: 5818
orientation: inline

ori
type: SequenceAnnotation
persistent identity: annotation2363687
display id: annotation2363687
version: 1
top level: BBa_K1160000
role: SO:0000110
role: feature/misc
location: https://synbiohub.org/public/igem/BBa_K1160000/annotation2363687/range2363687/1

https://synbiohub.org/public/igem/BBa_K1160000/annotation2363687/range2363687/1
type: Range
persistent identity: range2363687
display id: range2363687
version: 1
direction: direction/forward
top level: BBa_K1160000
start: 7404
end: 7992
orientation: inline

CMV enhancer
type: SequenceAnnotation
persistent identity: annotation2363683
display id: annotation2363683
version: 1
top level: BBa_K1160000
role: SO:0000110
role: feature/misc
location: https://synbiohub.org/public/igem/BBa_K1160000/annotation2363683/range2363683/1

https://synbiohub.org/public/igem/BBa_K1160000/annotation2363683/range2363683/1
type: Range
persistent identity: range2363683
display id: range2363683
version: 1
direction: direction/forward
top level: BBa_K1160000
start: 235
end: 614
orientation: inline

BSD
type: SequenceAnnotation
persistent identity: annotation2363686
display id: annotation2363686
version: 1
top level: BBa_K1160000
role: SO:0000110
role: feature/misc
location: https://synbiohub.org/public/igem/BBa_K1160000/annotation2363686/range2363686/1

https://synbiohub.org/public/igem/BBa_K1160000/annotation2363686/range2363686/1
type: Range
persistent identity: range2363686
display id: range2363686
version: 1
direction: direction/forward
top level: BBa_K1160000
start: 6276
end: 6644
orientation: inline

bGH poly(A) signal
type: SequenceAnnotation
persistent identity: annotation2363684
display id: annotation2363684
version: 1
top level: BBa_K1160000
role: SO:0000324
role: feature/tag
location: https://synbiohub.org/public/igem/BBa_K1160000/annotation2363684/range2363684/1

https://synbiohub.org/public/igem/BBa_K1160000/annotation2363684/range2363684/1
type: Range
persistent identity: range2363684
display id: range2363684
version: 1
direction: direction/forward
top level: BBa_K1160000
start: 5120
end: 5344
orientation: inline

CMV promoter
type: SequenceAnnotation
persistent identity: annotation2363682
display id: annotation2363682
version: 1
top level: BBa_K1160000
role: SO:0000167
role: feature/promoter
location: https://synbiohub.org/public/igem/BBa_K1160000/annotation2363682/range2363682/1

https://synbiohub.org/public/igem/BBa_K1160000/annotation2363682/range2363682/1
type: Range
persistent identity: range2363682
display id: range2363682
version: 1
direction: direction/forward
top level: BBa_K1160000
start: 615
end: 818
orientation: inline

BBa_K1179002
type: ComponentDefinition
persistent identity: BBa K1179002
display id: BBa_K1179002
version: 1
was generated by: https://synbiohub.org/public/igem/igem2sbol/1
description: Hef1A_Cas9-VP16
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modified: 2015-05-08T01:09:35Z
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