Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V006671 | lentiMPH v2 | 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
lentiMPH v2 is a lenti vector encoding the MS2-P65-HSF1 activator helper complex with a 2A Hygro resistance marker (EF1a-MS2-p65-HSF1-2A-Hygro-WPRE). This version has a higher virus titer than the first one.
- Vector Name:
- lentiMPH v2
- Antibiotic Resistance:
- Ampicillin
- Length:
- 11739 bp
- Type:
- Mammalian Expression, Lentiviral
- Replication origin:
- ori
- Selection Marker:
- Hygromycin
- Copy Number:
- High Copy
- Promoter:
- EF-1α core
- Cloning Method:
- Restriction Enzyme
- 5' Primer:
- cacatagcgtaaaaggagcaacatag
- 3' Primer:
- GTTTGGATCTTGGTTCATTCTCAAGCCTCAG
- Growth Strain(s):
- Stbl3
- Growth Temperature:
- 37℃
lentiMPH v2 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
- Joung J, Konermann S, Gootenberg JS, Abudayyeh OO, Platt RJ, Brigham MD, Sanjana NE, Zhang F. Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening. Nat Protoc. 2017 Apr;12(4):828-863.
lentiMPH v2 vector Sequence
LOCUS Exported 11739 bp ds-DNA circular SYN 20-DEC-2021
DEFINITION lenti vector encoding the MS2-P65-HSF1 activator helper complex with
a 2A Hygro resistance marker (EF1a-MS2-p65-HSF1-2A-Hygro-WPRE). This
version has a higher virus titer..
ACCESSION .
VERSION .
KEYWORDS lentiMPH v2
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 11739)
AUTHORS Joung J, Konermann S, Gootenberg JS, Abudayyeh OO, Platt RJ, Brigham
MD, Sanjana NE, Zhang F
TITLE Genome-scale CRISPR-Cas9 knockout and transcriptional activation
screening.
JOURNAL Nat Protoc. 2017 Apr;12(4):828-863. doi: 10.1038/nprot.2017.016.
Epub 2017 Mar 23.
PUBMED 28333914
REFERENCE 2 (bases 1 to 11739)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; doi:
"10.1038/nprot.2017.016"; journalName: "Nat Protoc"; date:
"2017-04"; volume: "12"; issue: "4"; pages: "828-863"
FEATURES Location/Qualifiers
source 1..11739
/organism="synthetic DNA construct"
/mol_type="other DNA"
primer_bind complement(85..105)
/label=pBABE 3'
/note="SV40 enhancer, reverse primer for pBABE vectors"
promoter 90..419
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
rep_origin 270..405
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind 332..351
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
promoter 467..514
/label=EM7 promoter
/note="synthetic bacterial promoter "
CDS 533..907
/codon_start=1
/gene="Sh ble from Streptoalloteichus hindustanus"
/product="antibiotic-binding protein"
/label=BleoR
/note="confers resistance to bleomycin, phleomycin, and
Zeocin(TM)"
/translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
EFALRDPAGNCVHFVAEEQD"
polyA_signal 1037..1158
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(1074..1093)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 1128..1147
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
primer_bind complement(1207..1223)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(1207..1223)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(1220..1242)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 1231..1247
/label=lac operator
/bound_moiety="lac repressor encoded by lacI"
/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(1255..1285)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 1300..1321
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(1438..1455)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(1609..2197)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(1689..1708)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(2368..3228)
/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 2991..3010
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(3229..3333)
/gene="bla"
/label=AmpR promoter
primer_bind complement(3408..3427)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
enhancer 3599..3978
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 3980..4178
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 4133..4153
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
LTR 4196..4376
/label=5' LTR (truncated)
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 4423..4548
/label=HIV-1 Psi
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 5041..5274
/label=RRE
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 5459..5503
/codon_start=1
/product="antigenic peptide corresponding to amino acids
655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik
et al., 2013)"
/label=gp41 peptide
/note="recognized by the 2H10 single-chain llama nanobody"
/translation="KNEQELLELDKWASL"
misc_feature 5801..5918
/label=cPPT/CTS
/note="central polypurine tract and central termination
sequence of HIV-1"
misc_feature 5971..6559
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
CDS 6077..6088
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/label=Factor Xa site
/translation="IEGR"
primer_bind 6486..6506
/label=WPRE-R
/note="WPRE, reverse primer"
CDS complement(6599..7618)
/codon_start=1
/product="hygromycin B phosphotransferase"
/label=HygR
/note="confers resistance to hygromycin"
/translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
"
CDS complement(7622..7675)
/codon_start=1
/product="2A peptide from Thosea asigna virus capsid
protein"
/label=T2A
/note="Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides."
/translation="EGRGSLLTCGDVEENPGP"
CDS complement(7691..8062)
/codon_start=1
/product="C-terminal activation domain from the human heat
shock transcription factor HSF1"
/label=HSF1
/translation="GFSVDTSALLDLFSPSVTVPDMSLPDLDSSLASIQELLSPQEPPR
PPEAENSSPDSGKQLVHYTAQPLFLLDPGSVDTGSNDLPVLFELGEGSYFSEGDGFAED
PTISLLTGSEPPKAKDPTVS"
CDS complement(8087..8629)
/codon_start=1
/product="C-terminal portion of the p65 subunit of mouse
NF-kappa-B"
/label=p65
/translation="PSGQISNQALALAPSSAPVLAQTMVPSSAMVPLAQPPAPAPVLTP
GPPQSLSAPVPKSTQAGEGTLSEALLHLQFDADEDLGALLGNSTDPGVFTDLASVDNSE
FQQLLNQGVSMSHSTAEPMLMEYPEAITRLVTGSQRPPDPAPTPLGTSGLPNGLSGDED
FSSIADMDFSALLSQISS"
CDS complement(8645..8665)
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS complement(8720..9106)
/codon_start=1
/product="bacteriophage MS2 coat protein"
/label=MS2-N55K
/note="binds with high affinity to a specific stem-loop
structure in the viral RNA (Lim et al., 1994)"
/translation="ASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVT
CSVRQSSAQKRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELI
VKAMQGLLKDGNPIPSAIAANSGIY"
regulatory 9106..9115
/regulatory_class="other"
/label=Kozak sequence
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
promoter complement(9122..10300)
/label=EF-1-alpha promoter
/note="strong constitutive promoter for human elongation
factor EF-1-alpha"
intron 9131..10069
/label=EF-1-alpha intron A
/note="intron upstream of the start codon of human
EF-1-alpha"
primer_bind complement(9154..9174)
/label=EF1a-F
/note="Human elongation factor-1a promoter, forward primer"
primer_bind complement(10383..10399)
/label=KS primer
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(10384..10400)
/label=pBluescriptKS
/note="For pBluescript vector"
LTR 10905..11085
/label=5' LTR (truncated)
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
primer_bind complement(11111..11128)
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer.
Also called BGH reverse"
polyA_signal 11117..11341
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
rep_origin join(11387..11739,1..76)
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(11474..11493)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 11684..11705
/label=F1ori-F
/note="F1 origin, forward primer"