Price Information
| Cat No. | Plasmid Name | Availability | Buy one, get one free! (?) |
|---|---|---|---|
| V017927 | lentiGuide-Hygro-EGFP | In stock, instant shipping |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
lentiGuide-Hygro-EGFP is a lentiviral vector for GNAS imprinting regions, delivering sgRNA, with hygromycin selection, EGFP tracing, aiding study of their long-range interactions to clarify pseudohypoparathyroidism type 1B pathogenesis.
- Vector Name:
- lentiGuide-Hygro-EGFP
- Antibiotic Resistance:
- Ampicillin
- Length:
- 11409 bp
- Type:
- Gene-editing vector
- Replication origin:
- ori
- Host:
- Mammalian cells, Lentivirus
- Selection Marker:
- Hyg
- Promoter:
- U6
- 5' Primer:
- PEF-F
- Growth Strain(s):
- Top10
- Growth Temperature:
- 30℃
lentiGuide-Hygro-EGFP 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
- Iwasaki Y, Aksu C, Reyes M, Ay B, He Q, Bastepe M. The long-range interaction between two GNAS imprinting control regions delineates pseudohypoparathyroidism type 1B pathogenesis. J Clin Invest. 2023 Apr 17;133(8):e167953. doi: 10.1172/JCI167953. PMID: 36853809; PMCID: PMC10104902.
lentiGuide-Hygro-EGFP vector Sequence
LOCUS . 11409 bp DNA circular UNK 01-JAN-1980
DEFINITION synthetic circular DNA.
ACCESSION
VERSION
KEYWORDS .
SOURCE .
ORGANISM .
.
FEATURES Location/Qualifiers
promoter 30..1208
/label="EF-1α promoter"
/note="strong constitutive promoter for human elongation
factor EF-1α"
CDS 1230..2249
/label="HygR"
/note="hygromycin B phosphotransferase"
CDS 2328..3044
/label="EGFP"
/note="the original enhanced GFP (Yang et al., 1996)"
misc_feature 3062..3650
/label="WPRE"
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
LTR 3722..3955
/label="3' LTR (ΔU3)"
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
polyA_signal 4033..4167
/label="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
rep_origin 4194..4329
/label="SV40 ori"
/note="SV40 origin of replication"
promoter complement(4350..4368)
/label="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
rep_origin 4536..4991
/label="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 5017..5121
/label="AmpR promoter"
CDS 5122..5979
/label="AmpR"
/note="β-lactamase"
rep_origin 6153..6741
/label="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
protein_bind 7029..7050
/label="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 7065..7095
/label="lac promoter"
/note="promoter for the E. coli lac operon"
promoter 7164..7182
/label="T3 promoter"
/note="promoter for bacteriophage T3 RNA polymerase"
promoter 7210..7436
/label="RSV promoter"
/note="Rous sarcoma virus enhancer/promoter"
misc_feature 7664..7789
/label="HIV-1 Ψ"
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 8282..8515
/label="RRE"
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 8700..8744
/label="gp41 peptide"
/note="antigenic peptide corresponding to amino acids 655
to 669 of the HIV envelope protein gp41 (Lutje Hulsik et
al., 2013)"
promoter 8922..9162
/label="U6 promoter"
/note="RNA polymerase III promoter for human U6 snRNA"
misc_RNA 11052..11127
/label="gRNA scaffold"
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
misc_feature 11183..11300
/label="cPPT/CTS"
/note="central polypurine tract and central termination
sequence of HIV-1"