M-SPcas vector (V012325)

MSDS

Basic Vector Information

- - Vector Name:
  - M-SPcas
- - Antibiotic Resistance:
  - Ampicillin
- - Length:
  - 9553 bp
- - Type:
  - CRISPR Plasmids
- - Replication origin:
  - ori
- - Source/Author:
  - Esvelt KM, Mali P, Braff JL, Moosburner M, Yaung SJ, Church GM.
- - Selection Marker:
  - Neomycin/G418(Geneticin)
- - Copy Number:
  - High copy number
- - Promoter:
  - SV40

M-SPcas vector Vector Map

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 ℃.

M-SPcas vector Sequence

Copy Sequence

Download GeneBank File(.gb)

LOCUS       M-SPcas.        9553 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Mammalian cell vector for expressing human codon-optimized 
            Streptococcus pyogenes Cas9 with a G418-resistance marker.
ACCESSION   .
VERSION     .
KEYWORDS    M-SPcas.
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9553)
  AUTHORS   Esvelt KM, Mali P, Braff JL, Moosburner M, Yaung SJ, Church GM.
  TITLE     Orthogonal Cas9 proteins for RNA-guided gene regulation and editing.
  JOURNAL   Nat. Methods 2013;10:1116-21.
  PUBMED    24076762
REFERENCE   2  (bases 1 to 9553)
  AUTHORS   Church Lab / Addgene #48668
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 9553)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat. 
            Methods"; date: "2013"; volume: "10"; pages: "1116-21"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..9553
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        50..429
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        430..633
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     CDS             748..4851
                     /label=Cas9
                     /note="Cas9 (Csn1) endonuclease from the Streptococcus
                     pyogenes Type II CRISPR/Cas system"
     CDS             4864..4884
                     /codon_start=1
                     /product="nuclear localization signal of SV40 large T
                     antigen"
                     /note="SV40 NLS"
                     /translation="PKKKRKV"
     polyA_signal    4983..5031
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5233..5661
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        5675..6004
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     CDS             6071..6862
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
     polyA_signal    7041..7174
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7211..7227)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(7235..7251)
                     /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(7259..7289)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(7304..7325)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(7613..8201)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(8375..9232)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(9233..9337)
                     /label=AmpR promoter

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