Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V000923 | pSH-EFIRES-B-AtAFB2-mCherry | 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:
- pSH-EFIRES-B-AtAFB2-mCherry
- Antibiotic Resistance:
- Ampicillin
- Length:
- 10442 bp
- Type:
- Mammalian Expression, CRISPR, TALEN ; Human safe h
- Replication origin:
- ori
- Selection Marker:
- Blasticidin ; mCherry for FACS enrichment
- Copy Number:
- High Copy
- Promoter:
- EF-1α
- Cloning Method:
- Restriction Enzyme
- 5' Primer:
- tctctccacaggtgtccact
- 3' Primer:
- acaccggccttattccaagc
pSH-EFIRES-B-AtAFB2-mCherry vector 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.
pSH-EFIRES-B-AtAFB2-mCherry vector Sequence
LOCUS Exported 10442 bp ds-DNA circular SYN 13-MAY-2021
DEFINITION AAVS1 safe harbor targeting vector with blasticidin resistance gene
expressing AtAFB2-mCherry.
ACCESSION .
VERSION .
KEYWORDS pSH-EFIRES-B-AtAFB2-mCherry
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 10442)
AUTHORS Li S, Prasanna X, Salo VT, Vattulainen I, Ikonen E
TITLE An efficient auxin-inducible degron system with low basal
degradation in human cells.
JOURNAL Nat Methods. 2019 Aug 26. pii: 10.1038/s41592-019-0512-x. doi:
10.1038/s41592-019-0512-x.
PUBMED 31451765
REFERENCE 2 (bases 1 to 10442)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Nat
Methods. 2019 Aug 26. pii: 10.1038/s41592-019-0512-x. doi:
10.1038/s41592-019-0512-x."
FEATURES Location/Qualifiers
source 1..10442
/organism="synthetic DNA construct"
/mol_type="other DNA"
primer_bind complement(135..152)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(306..894)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(386..405)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(1065..1925)
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
primer_bind 1688..1707
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(1926..2030)
/gene="bla"
/label=AmpR promoter
rep_origin 2057..2512
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(2144..2163)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 2354..2375
/label=F1ori-F
/note="F1 origin, forward primer"
misc_feature 3029..3832
/label=HA-L
/note="left homology arm from the adeno-associated virus
integration site (AAVS1) within intron 1 of the human
PPP1R12C gene"
polyA_signal 3858..3906
/note="synthetic polyadenylation signal"
misc_feature 3920..4011
/label=pause site
/note="RNA polymerase II transcriptional pause signal from
the human alpha-2 globin gene"
primer_bind 3960..3979
/label=RVprimer3
/note="pGL3 vector, forward primer"
promoter 4023..5185
/label=EF-1-alpha promoter
/note="strong constitutive promoter for human elongation
factor EF-1-alpha"
intron 4254..5176
/label=EF-1-alpha intron A
/note="intron upstream of the start codon of human
EF-1-alpha"
primer_bind 5133..5153
/label=EF1a-F
/note="Human elongation factor-1a promoter, forward primer"
intron 5224..5356
/label=chimeric intron
/note="chimera between introns from human beta-globin and
immunoglobulin heavy chain genes"
primer_bind 5339..5358
/label=pMT2-F
/note="Synthetic intron, forward primer"
promoter 5401..5419
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
CDS 7186..7893
/codon_start=1
/product="monomeric derivative of DsRed fluorescent protein
(Shaner et al., 2004)"
/label=mCherry
/note="mammalian codon-optimized"
/translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
GRHSTGGMDELYK"
primer_bind complement(7328..7346)
/label=mCherry-R
/note="mCherry, reverse primer"
primer_bind 7596..7615
/label=mCherry-F
/note="mCherry, forward primer"
misc_feature 7928..8480
/label=IRES
/note="internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)"
primer_bind complement(8072..8089)
/label=IRES reverse
/note="IRES internal ribosome entry site, reverse primer.
Also called pCDH-rev"
primer_bind 8299..8318
/label=IRES-F
/note="IRES internal ribosome entry site, forward primer"
CDS 8480..8881
/codon_start=1
/gene="Aspergillus terreus BSD"
/product="blasticidin S deaminase"
/label=BSD
/note="confers resistance to blasticidin"
/translation="MGAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFT
GVNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPG
IKAIVKDSDGQPTAVGIRELLPSGYVWEG"
polyA_signal 9039..9160
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(9076..9095)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 9130..9149
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
misc_feature 9196..10032
/label=HA-R
/note="right homology arm from the adeno-associated virus
integration site (AAVS1) within intron 1 of the human
PPP1R12C gene"