Price Information
| Cat No. | Plasmid Name | Availability | Buy one, get one free! (?) |
|---|---|---|---|
| V012838 | pLKO.1-EGFP-Puro | In stock, instant shipping |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
- Vector Name:
- pLKO.1-EGFP-Puro
- Antibiotic Resistance:
- Ampicillin
- Length:
- 8301 bp
- Type:
- Viral Expression & Packaging Vectors
- Replication origin:
- ori
- Copy Number:
- High copy number
- Promoter:
- hPGK
- Cloning Method:
- Enzyme Cut
- Growth Strain(s):
- DH5alpha
- Growth Temperature:
- 37℃
pLKO.1-EGFP-Puro 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.
pLKO.1-EGFP-Puro vector Sequence
LOCUS Exported 8301 bp DNA circular SYN 11-SEP-2025
DEFINITION Exported.
ACCESSION V012838
VERSION .
KEYWORDS pLKO.1-EGFP-Puro
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8301)
AUTHORS hfytfiu
TITLE Direct Submission
REFERENCE 2 (bases 1 to 8301)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..8301
/mol_type="other DNA"
/organism="synthetic DNA construct"
source join(2176..8301,1..2175)
/mol_type="other DNA"
/organism="synthetic DNA construct"
CDS 102..146
/label=gp41 peptide
/note="antigenic peptide corresponding to amino acids 655
to 669 of the HIV envelope protein gp41 (Lutje Hulsik et
al., 2013)"
CDS 295..336
/label=Protein Tat
/note="Protein Tat from Human immunodeficiency virus type 1
group M subtype B (isolate WMJ22). Accession#: P12509"
promoter 444..684
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA"
enhancer 706..1009
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 1010..1213
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
regulatory 1242..1251
/label=Kozak sequence
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
/regulatory_class="other"
CDS 1248..1967
/codon_start=1
/product="the original enhanced GFP (Yang et al., 1996)"
/label=EGFP
/note="mammalian codon-optimized"
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
EFVTAAGITLGMDELYK"
misc_feature 2006..2123
/label=cPPT/CTS
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 2172..2682
/label=hPGK promoter
/note="human phosphoglycerate kinase 1 promoter"
promoter 2172..2175
/label=hPGK promoter
/note="human phosphoglycerate kinase 1 promoter"
CDS 2704..3303
/codon_start=1
/gene="pac from Streptomyces alboniger"
/product="puromycin N-acetyltransferase"
/label=PuroR
/note="confers resistance to puromycin"
/translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
LTR 3431..3664
/label=3' LTR (Delta-U3)
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
polyA_signal 3736..3870
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
rep_origin 3897..4032
/label=SV40 ori
/note="SV40 origin of replication"
promoter complement(4053..4071)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(4081..4097)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
rep_origin 4239..4694
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 4720..4824
/label=AmpR promoter
CDS 4825..5685
/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"
rep_origin 5856..6444
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
protein_bind 6732..6753
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 6768..6798
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 6806..6822
/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 6830..6846
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
promoter 6867..6885
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
promoter 6913..7139
/label=RSV promoter
/note="Rous sarcoma virus enhancer/promoter"
LTR 7140..7320
/label=5' LTR (truncated)
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 7367..7492
/label=HIV-1 Psi
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 7985..8218
/label=RRE
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."