Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V011845 | pLSC-5 | 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:
- pLSC-5
- Antibiotic Resistance:
- Ampicillin
- Length:
- 13792 bp
- Type:
- Mammalian Expression, Lentiviral, CRISPR
- Replication origin:
- ori
- Selection Marker:
- Puromycin
- Promoter:
- U6
- Cloning Method:
- Restriction Enzyme
- 5' Primer:
- NA
pLSC-5 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.
pLSC-5 vector Sequence
LOCUS V011845 13792 bp DNA circular SYN 13-MAY-2021
DEFINITION Exported.
ACCESSION V011845
VERSION V011845
KEYWORDS pLSC-5
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
.
REFERENCE 1 (bases 1 to 13792)
AUTHORS Zetsche B, Volz SE, Zhang F
TITLE A split-Cas9 architecture for inducible genome editing and
transcription modulation.
JOURNAL Nat Biotechnol. 2015 Feb 2. doi: 10.1038/nbt.3149.
PUBMED 25643054
REFERENCE 2 (bases 1 to 13792)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 13792)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Nat
Biotechnol. 2015 Feb 2. doi: 10.1038/nbt.3149."
SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..13792
/mol_type="other DNA"
/organism="synthetic DNA construct"
primer_bind complement(47..66)
/label="pRS-marker"
/note="pRS vectors, use to sequence yeast selectable
marker"
enhancer 238..617
/label="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"
promoter 618..820
/label="CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
LTR 835..1015
/label="5' LTR (truncated)"
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 1062..1187
/label="HIV-1 Psi"
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 1680..1913
/label="RRE"
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 2098..2142
/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 2291..2332
/note="Protein Tat from Human immunodeficiency virus type 1
group M subtype B (isolate WMJ22). Accession#: P12509"
/label="Protein Tat"
misc_feature 2440..2557
/label="cPPT/CTS"
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 2608..2848
/label="U6 promoter"
/note="RNA polymerase III promoter for human U6 snRNA"
misc_RNA 2873..2948
/label="gRNA scaffold"
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
promoter 3010..3221
/label="EF-1-alpha core promoter"
/note="core promoter for human elongation factor
EF-1-alpha"
regulatory 3229..3238
/label="Kozak sequence"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
/regulatory_class="other"
CDS 3241..3261
/label="SV40 NLS"
/note="nuclear localization signal of SV40 (simian virus
40) large T antigen"
CDS 3286..3606
/label="FKBP"
/note="human FK506-binding protein FKBP12"
CDS 3652..6036
/label="Cas9(C)"
/note="C-terminal portion of Streptococcus pyogenes Cas9
(Zetsche et al., 2015)"
CDS 6037..6084
/label="nucleoplasmin NLS"
/note="bipartite nuclear localization signal from
nucleoplasmin"
CDS 6094..6150
/label="P2A"
/note="2A peptide from porcine teschovirus-1 polyprotein"
CDS 6172..7890
/label="Cas9(N)"
/note="N-terminal portion of Streptococcus pyogenes Cas9
(Zetsche et al., 2015)"
CDS 7936..8205
/label="FRB"
/note="FKBP-rapamycin binding domain of human FRAP"
CDS 8215..8271
/codon_start=1
/product="2A peptide from porcine teschovirus-1
polyprotein"
/label="P2A"
/note="Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides."
/translation="ATNFSLLKQAGDVEENPGP"
CDS 8272..8865
/label="PuroR"
/note="puromycin N-acetyltransferase"
misc_feature 8884..9472
/label="WPRE"
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
LTR 9544..9777
/label="3' LTR (Delta-U3)"
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
polyA_signal 9809..9920
/label="bGH poly(A) signal"
/note="bovine growth hormone polyadenylation signal"
rep_origin 10079..10507
/label="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 10521..10850
/label="SV40 promoter"
/note="SV40 enhancer and early promoter"
promoter 10898..10945
/label="EM7 promoter"
/note="synthetic bacterial promoter"
CDS 10964..11335
/label="BleoR"
/note="antibiotic-binding protein"
polyA_signal 11468..11601
/label="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
primer_bind complement(11638..11654)
/label="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(11638..11654)
/label="M13 Reverse"
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(11651..11673)
/label="M13/pUC Reverse"
/note="In lacZ gene"
protein_bind 11662..11678
/label="lac operator"
/bound_moiety="lac repressor encoded by lacI"
/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(11686..11716)
/label="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind complement(11731..11752)
/label="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(11869..11886)
/label="L4440"
/note="L4440 vector, forward primer"
rep_origin complement(12040..12628)
/direction=LEFT
/label="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(12802..13659)
/label="AmpR"
/note="beta-lactamase"
promoter complement(13660..13764)
/label="AmpR promoter"