pcDNA3.1-CibN-dCas9-CibN vector (V012279)

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LOCUS       pcDNA3.1-CibN-dC       10632 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Plasmid encoding two copies of CIBN fused to catalytically inactive 
            dCas9, for use in the light-activated CRISPR-Cas9 effector (LACE) 
            system.
ACCESSION   .
VERSION     .
KEYWORDS    pcDNA3.1-CibN-dCas9-CibN
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10632)
  AUTHORS   Polstein LR, Gersbach CA.
  TITLE     A light-inducible CRISPR-Cas9 system for control of endogenous gene 
            activation.
  JOURNAL   Nat. Chem. Biol. 2015;11:198-200.
  PUBMED    25664691
REFERENCE   2  (bases 1 to 10632)
  AUTHORS   Gersbach Lab / Addgene #60553
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 10632)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat. Chem. 
            Biol."; date: "2015"; volume: "11"; pages: "198-200"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..10632
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        421..750
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     CDS             817..1608
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
     polyA_signal    1785..1918
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        complement(2003..2033)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(2048..2069)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(2357..2942)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(3116..3973)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(3974..4078)
                     /label=AmpR promoter
     enhancer        4344..4723
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4724..4927
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     promoter        4972..4990
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             5013..5015
                     /codon_start=1
                     /product="start codon"
                     /label=start codon
                     /note="ATG"
                     /translation="M"
     CDS             5061..5570
                     /label=CIBN
                     /note="N-terminal portion of the CIB1 transcription factor
                     from Arabidopsis thaliana"
     CDS             5577..5600
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase
                     cleavage site"
                     /label=FLAG
                     /note="FLAG"
                     /translation="DYKDDDDK"
     CDS             5607..5627
                     /codon_start=1
                     /product="nuclear localization signal of SV40 large T
                     antigen"
                     /note="SV40 NLS"
                     /translation="PKKKRKV"
     CDS             5637..9740
                     /label=dCas9
                     /note="catalytically dead mutant of the Cas9 endonuclease
                     from the Streptococcus pyogenes Type II CRISPR/Cas system"
     CDS             9765..9785
                     /label=SV40 NLS
                     /note="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
     CDS             9798..10307
                     /codon_start=1
                     /product="N-terminal portion of the CIB1 transcription
                     factor from Arabidopsis thaliana"
                     /label=N-terminal portion of the CIB1 transcription
                     fa
                     /note="CIBN"
                     /note="binds to CRY2 (cryptochrome 2) that has absorbed
                     blue light (Kennedy et al., 2010)"
                     /translation="MNGAIGGDLLLNFPDMSVLERQRAHLKYLNPTFDSPLAGFFADSS
                     MITGGEMDSYLSTAGLNLPMMYGETTVEGDSRLSISPETTLGTGNFKKRKFDTETKDCN
                     EKKKKMTMNRDDLVEEGEEEKSKITEQNNGSTKSIKKMKHKAKKEENNFSNDSSKVTKE
                     LEKTDYI"
     polyA_signal    10341..10565
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"