Lenti-luciferase-P2A-Neo vector (V012116)

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V012116 Lenti-luciferase-P2A-Neo In stock, instant shipping

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Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.

Basic Vector Information

The Lenti-luciferase-P2A-Neo is a 3rd generation lentiviral plasmid for expression of luciferase with Neo resistance.

Vector Name:
Lenti-luciferase-P2A-Neo
Antibiotic Resistance:
Ampicillin
Length:
8833 bp
Type:
Mammalian Expression, Lentiviral, Luciferase
Replication origin:
ori
Selection Marker:
Neomycin (select with G418)
Promoter:
RSV
Cloning Method:
Ligation Independent Cloning
5' Primer:
AACCGGTGCCTAGAGAAGGT
Growth Strain(s):
Stbl3
Growth Temperature:
37℃

Lenti-luciferase-P2A-Neo vector Map

Lenti-luciferase-P2A-Neo8833 bp40080012001600200024002800320036004000440048005200560060006400680072007600800084008800RSV promoter5' LTR (truncated)HIV-1 PsiRREgp41 peptidecPPT/CTSEF-1-alpha core promoterluciferaseP2ANeoR/KanRWPRE3' LTR (Delta-U3)SV40 poly(A) signalSV40 oriT7 promoterM13 fwdf1 oriAmpR promoterAmpRoriL4440CAP binding sitelac promoterlac operatorM13 revT3 promoter

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

  • Xu Y, Milazzo JP, Somerville TDD, Tarumoto Y, Huang YH, Ostrander EL, Wilkinson JE, Challen GA, Vakoc CR. A TFIID-SAGA Perturbation that Targets MYB and Suppresses Acute Myeloid Leukemia. Cancer Cell. 2018 Jan 8;33(1):13-28.e8.

Lenti-luciferase-P2A-Neo vector Sequence

Copy Sequence

Download GenBank File(.gb)

LOCUS       40924_1659        8833 bp DNA     circular SYN 13-MAY-2021
DEFINITION  3rd generation lentiviral plasmid for expression of luciferase with 
            Neo resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8833)
  AUTHORS   Xu Y, Milazzo JP, Somerville TDD, Tarumoto Y, Huang YH, Ostrander 
            EL, Wilkinson JE, Challen GA, Vakoc CR
  TITLE     A TFIID-SAGA Perturbation that Targets MYB and Suppresses Acute 
            Myeloid Leukemia.
  JOURNAL   Cancer Cell. 2018 Jan 8;33(1):13-28.e8. doi: 
            10.1016/j.ccell.2017.12.002.
  PUBMED    29316427
REFERENCE   2  (bases 1 to 8833)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 8833)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.ccell.2017.12"; journalName: "Cancer Cell"; date: 
            "2018-01-8- 8"; volume: "33"; issue: "1"; pages: "13-28.e8"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..8833
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        5..231
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             232..412
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    459..584
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus
                     type 1"
     misc_feature    1077..1310
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1495..1539
                     /codon_start=1
                     /label=gp41 peptide
                     /note="antigenic peptide corresponding to amino acids 655
                     to 669 of the HIV envelope protein gp41 (Lutje Hulsik et 
                     al., 2013)"
                     /translation="KNEQELLELDKWASL"
     misc_feature    1723..1840
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination
                     sequence of HIV-1"
     promoter        1909..2120
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2146..3795
                     /codon_start=1
                     /label=luciferase
                     /note="firefly luciferase"
                     /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA
                     HIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP
                     ANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS
                     MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
                     RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK
                     IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY
                     GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS
                     GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL
                     LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV
                     FVDEVPKGLTGKLDARKIREILIKAKKGGKIAV"
     CDS             3805..3861
                     /codon_start=1
                     /label=P2A
                     /note="2A peptide from porcine teschovirus-1 polyprotein"
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             3880..4671
                     /codon_start=1
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     misc_feature    4690..5278
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional
                     regulatory element"
     LTR             5350..5583
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    5661..5795
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     rep_origin      5822..5957
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        complement(5978..5996)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6006..6022)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      6164..6619
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        6645..6749
                     /label=AmpR promoter
     CDS             6750..7607
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      7781..8369
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8523..8540
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    8657..8678
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        8693..8723
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8731..8747
                     /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)."
     primer_bind     8755..8771
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        8792..8810
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"