Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V013219 | pIRES-hrGFP-1a | In stock, instant shipping |
Buy one, get one free! |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
The plasmid pIRES-hrGFP-1a contains a multiple cloning site (MCS), an internal ribosome entry site (IRES), and codes for green fluorescent protein (hrGFP). It enables the expression of two genes independently from the same transcript and allows monitoring gene expression at the cellular level via GFP.
- Vector Name:
- pIRES-hrGFP-1a
- Antibiotic Resistance:
- Ampicillin
- Length:
- 4979 bp
- Type:
- Protein expression
- Replication origin:
- ori
- Host:
- Mammalian cells
- Promoter:
- CMV
- Growth Strain(s):
- stbl3
- Growth Temperature:
- 37℃
pIRES-hrGFP-1a 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.
References
- Oviedo N, Manuel-Apolinar L, Orozco-Suárez S, Juárez-Cedillo T, Bekker Méndez VC, Tesoro-Cruz E. Intranasal Administration of a Naked Plasmid Reached Brain Cells and Expressed Green Fluorescent Protein, a Candidate for Future Gene Therapy Studies. Arch Med Res. 2017 Oct;48(7):616-622. doi: 10.1016/j.arcmed.2018.03.003. Epub 2018 Mar 16. PMID: 29555303.
pIRES-hrGFP-1a vector Sequence
LOCUS 62056_12915 4979 bp DNA circular SYN 01-JAN-1980
DEFINITION synthetic circular DNA.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 4979)
AUTHORS .
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..4979
/mol_type="other DNA"
/organism="synthetic DNA construct"
CDS complement(4..861)
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 1031..1619
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
enhancer 1794..2097
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 2098..2301
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
promoter 2347..2365
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
CDS 2443..2514
/codon_start=1
/label=3xFLAG
/note="three tandem FLAG(R) epitope tags"
/translation="DYKDDDDKDYKDDDDKDYKDDDDK"
misc_feature 2550..3133
/label=IRES2
/note="internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)"
CDS 3134..3850
/codon_start=1
/label=hrGFP
/note="humanized Renilla green fluorescent protein"
/translation="MVSKQILKNTGLQEIMSFKVNLEGVVNNHVFTMEGCGKGNILFGN
QLVQIRVTKGAPLPFAFDILSPAFQYGNRTFTKYPEDISDFFIQSFPAGFVYERTLRYE
DGGLVEIRSDINLIEEMFVYRVEYKGRNFPNDGPVMKKTITGLQPSFEVVYMNDGVLVG
QVILVYRLNSGKFYSCHMRTLMKSKGVVKDFPEYHFIQHRLEKTYVEDGGFVEQHETAI
AQLTSLGKPLGSLHEWV"
promoter complement(3884..3902)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
polyA_signal 4176..4297
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
rep_origin complement(4304..4759)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 4786..4888
/label=AmpR promoter
protein_bind 4905..4938
/label=loxP
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (ATGTATGC) (Shaw et al., 2021)."