MLM3639 vector (V012321)

LOCUS       MLM3639.        7614 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Plasmid for expressing 3xFLAG(R)-tagged S. pyogenes Cas9 from either
            the CMV promoter in vivo or the T7 promoter in vitro.
ACCESSION   .
VERSION     .
KEYWORDS    MLM3639
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7614)
  AUTHORS   Hwang WY, Fu Y, Reyon D, Maeder ML, Tsai SQ, Sander JD, Peterson RT,
            Yeh JR, Joung JK.
  TITLE     Efficient genome editing in zebrafish using a CRISPR-Cas system.
  JOURNAL   Nat. Biotechnol. 2013;31:227-9.
  PUBMED    23360964
REFERENCE   2  (bases 1 to 7614)
  AUTHORS   Joung Lab / Addgene #42252
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 7614)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat. 
            Biotechnol."; date: "2013"; volume: "31"; pages: "227-9"
COMMENT     SGRef: number: 2; type: "Journal Article"
COMMENT     Linearize with PmeI for in vitro transcription.
FEATURES             Location/Qualifiers
     source          1..7614
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        132..335
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     promoter        377..395
                     /note="T7 promoter"
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             409..4512
                     /label=Cas9
                     /note="Cas9 (Csn1) endonuclease from the Streptococcus
                     pyogenes Type II CRISPR/Cas system"
     CDS             4519..4539
                     /codon_start=1
                     /product="nuclear localization signal of SV40 large T
                     antigen"
                     /note="SV40 NLS"
                     /translation="PKKKRKV"
     CDS             4546..4611
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=three tandem FLAG
                     /note="3xFLAG"
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             4629..4646
                     /label=6xHis
                     /note="6xHis affinity tag"
     polyA_signal    4675..4899
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4970..4986)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(4994..5010)
                     /label=lac operator
                     /note="The lac repressor binds to the lac operator to
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(5018..5048)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(5063..5084)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(5372..5960)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(6134..6991)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(6992..7096)
                     /label=AmpR promoter
     enhancer        7366..7614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"