MLM3639 vector (V012321)

Basic Vector Information

      • Vector Name:
      • MLM3639
      • Antibiotic Resistance:
      • Ampicillin
      • Length:
      • 7614 bp
      • Type:
      • CRISPR Plasmids
      • Source/Author:
      • Hwang WY, Fu Y, Reyon D, Maeder ML, Tsai SQ, Sander JD, Peterson RT,
      • Copy Number:
      • High copy number

MLM3639 vector Vector Map

MLM36397614 bp3006009001200150018002100240027003000330036003900420045004800510054005700600063006600690072007500CMV promoterT7 promoterCas9SV40 NLS3xFLAG6xHisbGH poly(A) signalM13 revlac operatorlac promoterCAP binding siteoriAmpRAmpR promoterCMV enhancer

Plasmid Resuspension Protocol:

1. Centrifuge at 5,000×g for 5 min.

2. Carefully open the tube and add 20 μl of sterile water to dissolve the DNA.

3. Close the tube and incubate for 10 minutes at room temperature.

4. Briefly vortex the tube and then do a quick spin to concentrate the liquid at the bottom. Speed is less than 5000×g.

5.Store the plasmid at -20 ℃.

MLM3639 vector Sequence

Copy Sequence

Download GeneBank File(.gb)

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"

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