Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V010684 | pC034 - LwCas13a-msfGFP-2A-Blast | 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:
- pC034 - LwCas13a-msfGFP-2A-Blast
- Antibiotic Resistance:
- Ampicillin
- Length:
- 13090 bp
- Type:
- Mammalian Expression, Lentiviral
- Replication origin:
- ori
- Selection Marker:
- Blasticidin
- Copy Number:
- High Copy
- Promoter:
- EFS
- Cloning Method:
- Gibson Cloning
- 5' Primer:
- ctaggtcttgaaaggagtggg
pC034 - LwCas13a-msfGFP-2A-Blast 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.
pC034 - LwCas13a-msfGFP-2A-Blast vector Sequence
LOCUS V010684 13090 bp DNA circular SYN 13-MAY-2021
DEFINITION Exported.
ACCESSION V010684
VERSION V010684
KEYWORDS pC034 - LwCas13a-msfGFP-2A-Blast
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
.
REFERENCE 1 (bases 1 to 13090)
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 13090)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 13090)
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..13090
/mol_type="other DNA"
/organism="synthetic DNA construct"
enhancer 1..380
/label="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"
promoter 381..583
/label="CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
LTR 598..778
/label="5' LTR (truncated)"
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 825..950
/label="HIV-1 Psi"
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 1443..1676
/label="RRE"
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 1861..1905
/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 2054..2095
/note="Protein Tat from Human immunodeficiency virus type 1
group M subtype B (isolate WMJ22). Accession#: P12509"
/label="Protein Tat"
misc_feature 2203..2320
/label="cPPT/CTS"
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 2413..2624
/label="EF-1-alpha core promoter"
/note="core promoter for human elongation factor
EF-1-alpha"
CDS 2652..2699
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label="nucleoplasmin NLS"
/translation="KRPAATKKAGQAKKKK"
CDS 2706..6161
/label="LwCas13a"
/note="Leptotrichia wadei CRISPR-Cas effector that acts as
an RNA-guided RNAse (Abudayyeh et al., 2017)"
CDS 6207..6920
/label="superfolder GFP"
/note="GFP variant that folds robustly even when fused to
poorly folded proteins (Pedelacq et al., 2006)"
CDS 6927..6974
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label="nucleoplasmin NLS"
/translation="KRPAATKKAGQAKKKK"
CDS 6975..7001
/label="HA"
/note="HA (human influenza hemagglutinin) epitope tag"
CDS 7002..7028
/label="HA"
/note="HA (human influenza hemagglutinin) epitope tag"
CDS 7029..7055
/label="HA"
/note="HA (human influenza hemagglutinin) epitope tag"
CDS 7065..7121
/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 7122..7520
/codon_start=1
/gene="Aspergillus terreus BSD"
/product="blasticidin S deaminase"
/label="BSD"
/note="confers resistance to blasticidin"
/translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
KAIVKDSDGQPTAVGIRELLPSGYVWEG"
misc_feature 7545..8133
/label="WPRE"
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
primer_bind complement(8136..8152)
/label="KS primer"
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(8137..8153)
/label="pBluescriptKS"
/note="For pBluescript vector"
LTR 8658..8838
/label="5' LTR (truncated)"
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
polyA_signal 8870..9094
/label="bGH poly(A) signal"
/note="bovine growth hormone polyadenylation signal"
rep_origin 9140..9568
/label="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 9582..9911
/label="SV40 promoter"
/note="SV40 enhancer and early promoter"
promoter 9959..10006
/label="EM7 promoter"
/note="synthetic bacterial promoter "
CDS 10025..10396
/label="BleoR"
/note="antibiotic-binding protein"
polyA_signal 10529..10662
/label="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
primer_bind complement(10699..10715)
/label="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(10699..10715)
/label="M13 Reverse"
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(10712..10734)
/label="M13/pUC Reverse"
/note="In lacZ gene"
protein_bind 10723..10739
/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(10747..10777)
/label="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind complement(10792..10813)
/label="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(10930..10947)
/label="L4440"
/note="L4440 vector, forward primer"
rep_origin complement(11101..11689)
/direction=LEFT
/label="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(11863..12720)
/label="AmpR"
/note="beta-lactamase"
promoter complement(12721..12825)
/label="AmpR promoter"
primer_bind complement(12900..12919)
/label="pRS-marker"
/note="pRS vectors, use to sequence yeast selectable
marker"