Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V012162 | pirespuro2-CTLA4-mEos2 | 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:
- pirespuro2-CTLA4-mEos2
- Antibiotic Resistance:
- Ampicillin
- Length:
- 6485 bp
- Type:
- Mammalian Expression
- Replication origin:
- ori
- Copy Number:
- High Copy
- Promoter:
- CMV
- Cloning Method:
- Restriction Enzyme
- 5' Primer:
- CMV-F
pirespuro2-CTLA4-mEos2 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.
pirespuro2-CTLA4-mEos2 vector Sequence
LOCUS 40924_25711 6485 bp DNA circular SYN 13-MAY-2021
DEFINITION mammalian expression of CTLA4-mEos2.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6485)
AUTHORS Fricke F, Beaudouin J, Eils R, Heilemann M
TITLE One, two or three? Probing the stoichiometry of membrane proteins by
single-molecule localization microscopy.
JOURNAL Sci Rep. 2015 Sep 11;5:14072. doi: 10.1038/srep14072.
PUBMED 26358640
REFERENCE 2 (bases 1 to 6485)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 6485)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Sci Rep.";
date: "2015-09-11"; pages: "
10.1038/srep14072"
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..6485
/mol_type="other DNA"
/organism="synthetic DNA construct"
enhancer 11..390
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 391..594
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
promoter 639..657
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
regulatory 709..718
/label=Kozak sequence
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
/regulatory_class="other"
CDS 1378..2055
/codon_start=1
/label=mEos2
/note="monomeric variant of green-to-red photoswitchable
fluorescent protein EosFP (McKinney et al., 2009)"
/translation="MSAIKPDMKIKLRMEGNVNGHHFVIDGDGTGKPFEGKQSMDLEVK
EGGPLPFAFDILTTAFHYGNRVFAKYPDNIQDYFKQSFPKGYSWERSLTFEDGGICIAR
NDITMEGDTFYNKVRFYGTNFPANGPVMQKKTLKWEPSTEKMYVRDGVLTGDIHMALLL
EGNAHYRCDFRTTYKAKEKGVKLPGYHFVDHCIEILSHDKDYNKVKLYEHAVAHSGLPD
NARR"
intron 2128..2357
/label=chimeric intron
/note="chimera between introns from adenovirus and
immunoglobulin heavy chain genes"
misc_feature 2419..2980
/label=IRES
/note="internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)"
CDS 3013..3609
/codon_start=1
/label=PuroR
/note="puromycin N-acetyltransferase"
/translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
polyA_signal 3754..3978
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
primer_bind 4025..4044
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
primer_bind complement(4104..4120)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
protein_bind complement(4128..4144)
/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(4152..4182)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind complement(4197..4218)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(4335..4352)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(4506..5094)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(5268..6125)
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(6126..6230)
/label=AmpR promoter
primer_bind complement(6305..6324)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"