Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V006881 | pTet-O-Ngn2-puro | 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:
- pTet-O-Ngn2-puro
- Antibiotic Resistance:
- Ampicillin
- Length:
- 9886 bp
- Type:
- Mammalian Expression, Lentiviral
- Replication origin:
- ori
- Selection Marker:
- Puromycin
- Copy Number:
- High Copy
- Promoter:
- EM7
- Cloning Method:
- Restriction Enzyme
- 5' Primer:
- LNCX
- 3' Primer:
- WPRE-R
pTet-O-Ngn2-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.
pTet-O-Ngn2-puro vector Sequence
LOCUS V006881 9886 bp DNA circular SYN 13-MAY-2021
DEFINITION Exported.
ACCESSION V006881
VERSION V006881
KEYWORDS pTet-O-Ngn2-puro
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
.
REFERENCE 1 (bases 1 to 9886)
AUTHORS Zhang Y, Pak C, Han Y, Ahlenius H, Zhang Z, Chanda S, Marro S,
Patzke C, Acuna C, Covy J, Xu W, Yang N, Danko T, Chen L, Wernig M,
Sudhof TC
TITLE Rapid single-step induction of functional neurons from human
pluripotent stem cells.
JOURNAL Neuron. 2013 Jun 5;78(5):785-98. doi: 10.1016/j.neuron.2013.05.029.
PUBMED 23764284
REFERENCE 2 (bases 1 to 9886)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 9886)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; doi:
"10.1016/j.neuron.2013.05"; journalName: "Neuron"; date: "2013-06-5-
5"; volume: "78"; issue: "5"; pages: "785-98"
SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..9886
/mol_type="other DNA"
/organism="synthetic DNA construct"
enhancer 100..479
/label="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"
promoter 480..682
/label="CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
LTR 697..877
/label="5' LTR (truncated)"
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 924..1049
/label="HIV-1 Psi"
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 1542..1775
/label="RRE"
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 1960..2004
/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 2153..2194
/note="Protein Tat from Human immunodeficiency virus type 1
group M subtype B (isolate WMJ22). Accession#: P12509"
/label="Protein Tat"
misc_feature 2302..2419
/label="cPPT/CTS"
/note="central polypurine tract and central termination
sequence of HIV-1"
protein_bind 2476..2746
/label="tetracycline response element"
/note="contains seven copies of the tetracycline operator
tetO"
promoter 2779..2817
/label="minimal CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 2814..2838
/label="LNCX"
/note="Human CMV promoter, forward primer"
regulatory 2917..2926
/label="Kozak sequence"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
/regulatory_class="other"
CDS 2926..2949
/label="FLAG"
/note="FLAG(R) epitope tag, followed by an enterokinase
cleavage site"
CDS 2950..3735
/gene="Neurog2"
/label="Neurogenin-2"
/note="Neurogenin-2 from Mus musculus. Accession#: P70447"
CDS 3742..3795
/label="T2A"
/note="2A peptide from Thosea asigna virus capsid protein"
CDS 3802..4398
/label="PuroR"
/note="puromycin N-acetyltransferase"
misc_feature 4440..5028
/label="WPRE"
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
primer_bind complement(5031..5047)
/label="KS primer"
/note="common sequencing primer, one of multiple similar
variants"
LTR 5553..5733
/label="5' LTR (truncated)"
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
polyA_signal 5765..5989
/label="bGH poly(A) signal"
/note="bovine growth hormone polyadenylation signal"
rep_origin 6035..6463
/label="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 6477..6806
/label="SV40 promoter"
/note="SV40 enhancer and early promoter"
promoter 6854..6901
/label="EM7 promoter"
/note="synthetic bacterial promoter"
CDS 6920..7291
/label="BleoR"
/note="antibiotic-binding protein"
polyA_signal 7424..7557
/label="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
primer_bind complement(7594..7610)
/label="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
protein_bind complement(7618..7634)
/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)."
promoter complement(7642..7672)
/label="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind complement(7687..7708)
/label="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(7825..7842)
/label="L4440"
/note="L4440 vector, forward primer"
rep_origin complement(7996..8584)
/direction=LEFT
/label="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(8758..9615)
/label="AmpR"
/note="beta-lactamase"
promoter complement(9616..9720)
/label="AmpR promoter"
primer_bind complement(9795..9814)
/label="pRS-marker"
/note="pRS vectors, use to sequence yeast selectable
marker"