Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V000006 | pHAGE-IRES-puro-NLS-dPspCas13b-EGFP-NLS-3xFlag | 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:
- pHAGE-IRES-puro-NLS-dPspCas13b-EGFP-NLS-3xFlag
- Antibiotic Resistance:
- Ampicillin
- Length:
- 12449 bp
- Type:
- Mammalian Expression, Lentiviral
- Replication origin:
- ori
- Copy Number:
- High Copy
- Promoter:
- EF-1α
- Cloning Method:
- Ligation Independent Cloning
pHAGE-IRES-puro-NLS-dPspCas13b-EGFP-NLS-3xFlag 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.
pHAGE-IRES-puro-NLS-dPspCas13b-EGFP-NLS-3xFlag vector Sequence
LOCUS V000006 12449 bp DNA circular SYN 13-MAY-2021
DEFINITION Exported.
ACCESSION V000006
VERSION V000006
KEYWORDS pHAGE-IRES-puro-NLS-dPspCas13b-EGFP-NLS-3xFlag
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
.
REFERENCE 1 (bases 1 to 12449)
AUTHORS Liang-Zhong Yang, Yang Wang, Si-Qi Li, Huang Wu, Gordon G.
Carmichael, Ling-Ling Chen
TITLE dCas13 imaging
JOURNAL Molecular Cell
REFERENCE 2 (bases 1 to 12449)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 12449)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Molecular
Cell"
SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..12449
/mol_type="other DNA"
/organism="synthetic DNA construct"
LTR 1..634
/label="3' LTR"
/note="3' long terminal repeat (LTR) from HIV-1"
misc_feature 681..806
/label="HIV-1 Psi"
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 1303..1536
/label="RRE"
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 1721..1765
/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 1914..1955
/note="Protein Tat from Human immunodeficiency virus type 1
group M subtype B (isolate WMJ22). Accession#: P12509"
/label="Protein Tat"
misc_feature 2058..2175
/label="cPPT/CTS"
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 2223..3401
/label="EF-1-alpha promoter"
/note="strong constitutive promoter for human elongation
factor EF-1-alpha"
CDS 3431..3451
/label="SV40 NLS"
/note="nuclear localization signal of SV40 (simian virus
40) large T antigen"
CDS 6752..7468
/label="EGFP"
/note="enhanced GFP"
CDS 7496..7543
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label="nucleoplasmin NLS"
/translation="KRPAATKKAGQAKKKK"
CDS 7604..7627
/label="FLAG"
/note="FLAG(R) epitope tag, followed by an enterokinase
cleavage site"
misc_feature 7634..8201
/label="IRES"
/note="internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)"
CDS 8221..8817
/label="PuroR"
/note="puromycin N-acetyltransferase"
misc_feature 8839..9427
/label="WPRE"
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
LTR 9502..9735
/label="3' LTR (Delta-U3)"
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
primer_bind complement(9854..9872)
/label="pBRforEco"
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 9940..10044
/gene="bla"
/label="AmpR promoter"
CDS 10045..10902
/label="AmpR"
/note="beta-lactamase"
rep_origin 11076..11664
/label="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 11818..11835
/label="L4440"
/note="L4440 vector, forward primer"
primer_bind 11927..11946
/label="SV40pro-F"
/note="SV40 promoter/origin, forward primer"
protein_bind 12075..12096
/label="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 12111..12141
/label="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind 12149..12165
/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 12173..12189
/label="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
primer_bind 12239..12258
/label="EBV-rev"
/note="SV40 polyA terminator, reverse primer"