Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V007161 | pENTR5'_ubi:loxP-EGFP-loxP | In stock, 1 week for quality controls |
Buy one, get one free! |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
- Vector Name:
- pENTR5'_ubi:loxP-EGFP-loxP
- Antibiotic Resistance:
- Kanamycin
- Length:
- 7496 bp
- Type:
- Multisite Gateway 5' entry vector
- Replication origin:
- ori
- Copy Number:
- High Copy
- Promoter:
- Ubi
- Cloning Method:
- Gateway Cloning
- 5' Primer:
- M13 forward
- 3' Primer:
- M13 reverse
pENTR5'_ubi:loxP-EGFP-loxP vector Map
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.
pENTR5'_ubi:loxP-EGFP-loxP vector Sequence
LOCUS 40924_17537 7496 bp DNA circular SYN 13-MAY-2021
DEFINITION synthetic circular DNA.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7496)
AUTHORS Mosimann C, Kaufman CK, Li P, Pugach EK, Tamplin OJ, Zon LI
TITLE Ubiquitous transgene expression and Cre-based recombination driven
by the ubiquitin promoter in zebrafish.
JOURNAL Development. 2011 Jan . 138(1):169-77.
PUBMED 21138979
REFERENCE 2 (bases 1 to 7496)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 7496)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName:
"Development. 2011 Jan . 138(1):169-77."
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..7496
/mol_type="other DNA"
/organism="synthetic DNA construct"
primer_bind 92..109
/label=L4440
/note="L4440 vector, forward primer"
terminator complement(268..295)
/label=rrnB T2 terminator
/note="transcription terminator T2 from the E. coli rrnB
gene"
terminator complement(387..473)
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"
primer_bind 537..553
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
protein_bind 592..691
/label=attL1
/note="recombination site for the Gateway(R) LR reaction"
promoter 715..4197
/label=Ubi
/note="Zebrafish ubiquitin (ubi) promoter"
protein_bind 4210..4243
/label=loxP
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT)."
CDS 4256..4972
/codon_start=1
/label=EGFP
/note="enhanced GFP"
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
EFVTAAGITLGMDELYK"
polyA_signal 5098..5219
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(5363..5384)
/label=F1ori-F
/note="F1 origin, forward primer"
protein_bind complement(5473..5506)
/label=loxP
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (ATGTATGC) (Shaw et al., 2021)."
protein_bind 5537..5661
/label=attR1
/note="recombination site for the Gateway(R) LR reaction"
promoter complement(5738..5756)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(5761..5777)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
CDS 5890..6696
/codon_start=1
/label=KanR
/note="aminoglycoside phosphotransferase"
/translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
rep_origin 6846..7434
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"