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p415-GalL-Cas9-CYC1t vector (V012313)

Price Information

Cat No. Plasmid Name Availability Add to cart
V012313 p415-GalL-Cas9-CYC1t In stock, 1 week for quality controls

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Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.

Basic Vector Information

Vector Name:
p415-GalL-Cas9-CYC1t
Antibiotic Resistance:
Ampicillin
Length:
10793 bp
Type:
CRISPR Plasmids
Replication origin:
ori
Source/Author:
DiCarlo JE, Norville JE, Mali P, Rios X, Aach J, Church GM.
Copy Number:
High copy number
Promoter:
GAL1, T3

p415-GalL-Cas9-CYC1t vector Map

Copy Sequence View Map
p415-GalL-Cas9-CYC1t10793 bp5001000150020002500300035004000450050005500600065007000750080008500900095001000010500CEN/ARSAmpR promoterAmpRoriCAP binding sitelac promoterT3 promoterGAL1 promoterCas9SV40 NLSCYC1 terminatorT7 promoterf1 oriLEU2 promoterLEU2

Plasmid 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 ℃.

6. The concentration of plasmid re-measurement sometimes differs from the nominal value, which may be due to the position of the lyophilized plasmid in the tube, the efficiency of the re-dissolution, the measurement bias, and adsorption on the wall of the tube, therefore, it is recommended to transform and extract the plasmid before using it

General Plasmid Transform Protocol

1. Take one 100μl of the competent cells and thaw it on ice for 10min, add 2μl of plasmid, then ice bath for 30min, then heat-shock it at 42℃ for 60s, do not stir, and then ice bath for 2min.

2. Add 900μl of LB liquid medium without antibiotics, and incubate at 37℃ for 45min (30℃ for 1-1.5 hours) with 180rpm shaking.

3. Centrifuge at 6000rpm for 5min, leave only 100μl of supernatant to resuspend the bacterial precipitate and spread it onto the target plasmid-resistant LB plate.

4. Invert the plate and incubate at 37℃ for 14h, or at 30℃ for 20h.

5. Pick a single colony into LB liquid medium, add the corresponding antibiotics, incubate at 220rpm for 14h, and extract the plasmid according to the experimental needs and the instructions of the plasmid extraction kit.

p415-GalL-Cas9-CYC1t vector Sequence

LOCUS       p415-GalL-Cas9-C       10793 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Yeast centromeric plasmid for expressing human codon-optimized Cas9 
            with the inducible GAL1 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    p415-GalL-Cas9-CYC1t
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10793)
  AUTHORS   DiCarlo JE, Norville JE, Mali P, Rios X, Aach J, Church GM.
  TITLE     Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas 
            systems.
  JOURNAL   Nucleic Acids Res. 2013;41:4336-43.
  PUBMED    23460208
REFERENCE   2  (bases 1 to 10793)
  AUTHORS   Church Lab / Addgene #43804
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 10793)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nucleic 
            Acids Res."; date: "2013"; volume: "41"; pages: "4336-43"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..10793
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    complement(70..573)
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an
                     autonomously replicating sequence"
     promoter        610..714
                     /label=AmpR promoter
     CDS             715..1572
                     /label=AmpR
                     /note="beta-lactamase"
     rep_origin      1746..2334
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     protein_bind    2622..2643
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        2658..2688
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     promoter        2757..2775
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        2794..3220
                     /gene="S. cerevisiae GAL1"
                     /label=S. cerevisiae GAL1 promoter
                     /note="GAL1 promoter"
                     /note="inducible promoter, regulated by Gal4"
     CDS             3258..7361
                     /label=Cas9
                     /note="Cas9 (Csn1) endonuclease from the Streptococcus
                     pyogenes Type II CRISPR/Cas system"
     CDS             7374..7394
                     /codon_start=1
                     /product="nuclear localization signal of SV40 large T
                     antigen"
                     /note="SV40 NLS"
                     /translation="PKKKRKV"
     terminator      7410..7657
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     promoter        complement(7676..7694)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     rep_origin      7865..8320
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        8620..9024
                     /label=LEU2 promoter
     CDS             9037..10128
                     /label=LEU2
                     /note="3-isopropylmalate dehydrogenase, required for
                     leucine biosynthesis"