pCAG-Cre vector (Cat. No.: V012688)

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Basic Information

Note: Mammalian expression of Cre recombinase from the broadly active CAG promoter/enhancer.

Name:: pCAG-Cre

Antibiotic Resistance:: Ampicillin

Length:: 5871 bp

Type:: CRE/LOXP

Replication origin:: ori

Source/Author:: Dr. Connie Cepko's lab

Copy Number:: High copy number

Promoter:: chicken β-actin

Growth Strain(s):: DH5alpha

Growth Temperature:: 37℃

Expression Method:: Transient

$ 198.2

In stock, 1 week for quality controls

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

Resources

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.

References

Matsuda T, Cepko CL. Controlled expression of transgenes introduced by in vivo electroporation. Proc Natl Acad Sci U S A. 2007 Jan 16;104(3):1027-32. doi: 10.1073/pnas.0610155104. Epub 2007 Jan 5. PMID: 17209010; PMCID: PMC1764220.

pCAG-Cre vector (Cat. No.: V012688) Sequence

LOCUS       pCAG-Cre.        5871 bp DNA     circular SYN 06-APR-2021
DEFINITION  synthetic circular DNA.
ACCESSION   .
VERSION     .
KEYWORDS    pCAG-Cre
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5871)
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 5871)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..5871
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        4..383
                     /note="CMV enhancer"
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        386..661
                     /note="chicken beta-actin promoter"
     intron          662..1670
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and
                     rabbit beta-globin"
     CDS             1737..2765
                     /label=Cre
                     /note="site-specific recombinase"
     CDS             2775..2804
                     /label=Myc
                     /note="Myc (human c-Myc proto-oncogene) epitope tag"
     polyA_signal    2960..3015
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3374..3390)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(3398..3414)
                     /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(3422..3452)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(3467..3488)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        3546..3742
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     polyA_signal    3748..3882
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     rep_origin      complement(4121..4709)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4883..5740)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(5741..5845)
                     /label=AmpR promoter