Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V012226 | pC014 - LwCas13a-msfGFP | 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:
- pC014 - LwCas13a-msfGFP
- Antibiotic Resistance:
- Ampicillin
- Length:
- 13585 bp
- Type:
- Mammalian Expression
- Replication origin:
- ori
- Copy Number:
- High Copy
- Promoter:
- EF-1α
- Cloning Method:
- Gibson Cloning
- 5' Primer:
- EF-1a Forward
- 3' Primer:
- WPRE-R
pC014 - LwCas13a-msfGFP 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.
pC014 - LwCas13a-msfGFP vector Sequence
LOCUS V012226 13585 bp DNA circular SYN 13-MAY-2021
DEFINITION Exported.
ACCESSION V012226
VERSION V012226
KEYWORDS pC014 - LwCas13a-msfGFP
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
.
REFERENCE 1 (bases 1 to 13585)
AUTHORS Abudayyeh OO, Gootenberg JS, Essletzbichler P, Han S, Joung J,
Belanto JJ, Verdine V, Cox DBT, Kellner MJ, Regev A, Lander ES,
Voytas DF, Ting AY, Zhang F
TITLE RNA targeting with CRISPR-Cas13.
JOURNAL Nature. 2017 Oct 4. doi: 10.1038/nature24049.
PUBMED 28976959
REFERENCE 2 (bases 1 to 13585)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 13585)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Nature.
2017 Oct 4. doi: 10.1038/nature24049."
SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..13585
/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 2682..3860
/label="EF-1-alpha promoter"
/note="strong constitutive promoter for human elongation
factor EF-1-alpha"
regulatory 3867..3876
/label="Kozak sequence"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
/regulatory_class="other"
CDS 3882..3902
/label="SV40 NLS"
/note="nuclear localization signal of SV40 (simian virus
40) large T antigen"
CDS 3909..7364
/label="LwCas13a"
/note="Leptotrichia wadei CRISPR-Cas effector that acts as
an RNA-guided RNAse (Abudayyeh et al., 2017)"
CDS 7410..8123
/label="superfolder GFP"
/note="GFP variant that folds robustly even when fused to
poorly folded proteins (Pedelacq et al., 2006)"
CDS 8139..8159
/label="SV40 NLS"
/note="nuclear localization signal of SV40 (simian virus
40) large T antigen"
CDS 8169..8195
/label="HA"
/note="HA (human influenza hemagglutinin) epitope tag"
CDS 8196..8222
/label="HA"
/note="HA (human influenza hemagglutinin) epitope tag"
CDS 8223..8249
/label="HA"
/note="HA (human influenza hemagglutinin) epitope tag"
misc_feature 8277..8865
/label="WPRE"
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
primer_bind complement(8868..8884)
/label="KS primer"
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(8869..8885)
/label="pBluescriptKS"
/note="For pBluescript vector"
LTR 9390..9570
/label="5' LTR (truncated)"
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
polyA_signal 9602..9826
/label="bGH poly(A) signal"
/note="bovine growth hormone polyadenylation signal"
rep_origin 9872..10300
/direction=RIGHT
/label="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(9959..9978)
/label="F1ori-R"
/note="F1 origin, reverse primer"
primer_bind 10169..10190
/label="F1ori-F"
/note="F1 origin, forward primer"
promoter 10314..10643
/label="SV40 promoter"
/note="SV40 enhancer and early promoter"
promoter 10691..10738
/label="EM7 promoter"
/note="synthetic bacterial promoter"
CDS 10757..11128
/label="BleoR"
/note="antibiotic-binding protein"
polyA_signal 11261..11394
/label="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
primer_bind complement(11431..11447)
/label="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(11431..11447)
/label="M13 Reverse"
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(11444..11466)
/label="M13/pUC Reverse"
/note="In lacZ gene"
protein_bind 11455..11471
/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(11479..11509)
/label="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind complement(11524..11545)
/label="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(11662..11679)
/label="L4440"
/note="L4440 vector, forward primer"
rep_origin complement(11833..12421)
/direction=LEFT
/label="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(12595..13452)
/label="AmpR"
/note="beta-lactamase"
promoter complement(13453..13557)
/label="AmpR promoter"