Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V000708 | pRDA_256 | 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:
- pRDA_256
- Antibiotic Resistance:
- Ampicillin
- Length:
- 14128 bp
- Type:
- Mammalian Expression, Lentiviral
- Replication origin:
- ori
- Copy Number:
- High Copy
- Promoter:
- SV40
pRDA_256 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.
pRDA_256 vector Sequence
LOCUS V000708 14128 bp DNA circular SYN 13-MAY-2021
DEFINITION Exported.
ACCESSION V000708
VERSION V000708
KEYWORDS pRDA_256
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
.
REFERENCE 1 (bases 1 to 14128)
AUTHORS Hanna RE, Hegde M, Fagre CR, DeWeirdt PC, Sangree AK, Szegletes Z,
Griffith A, Feeley MN, Sanson KR, Baidi Y, Koblan LW, Liu DR, Neal
JT, Doench JG
TITLE Massively parallel assessment of human variants with base editor
screens
JOURNAL BioRxiv
REFERENCE 2 (bases 1 to 14128)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 14128)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "BioRxiv"
SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..14128
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter 58..156
/label="CMVd1 promoter"
/note="truncated human cytomegalovirus (CMV) immediate
early promoter that yields slightly reduced expression
relative to the full-length promoter"
LTR 171..351
/label="5' LTR (truncated)"
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 398..523
/label="HIV-1 Psi"
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 1016..1249
/label="RRE"
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 1434..1478
/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 1627..1668
/note="Protein Tat from Human immunodeficiency virus type 1
group M subtype B (isolate WMJ22). Accession#: P12509"
/label="Protein Tat"
misc_feature 1776..1893
/label="cPPT/CTS"
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 1944..2184
/label="U6 promoter"
/note="RNA polymerase III promoter for human U6 snRNA"
misc_RNA 2214..2289
/label="gRNA scaffold"
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
promoter 2373..2584
/label="EF-1-alpha core promoter"
/note="core promoter for human elongation factor
EF-1-alpha"
CDS 2634..2654
/label="SV40 NLS"
/note="nuclear localization signal of SV40 (simian virus
40) large T antigen"
CDS 2652..3338
/label="APOBEC-1"
/note="cytidine deaminase (C to U editing enzyme) from rat"
CDS 3435..7535
/label="Cas9(D10A)"
/note="nickase mutant of the Cas9 endonuclease from the
Streptococcus pyogenes Type II CRISPR/Cas system"
CDS 7566..7814
/label="UGI"
/note="uracil-DNA glycosylase inhibitor from a Bacillus
subtilis bacteriophage (Mol et al., 1995)"
CDS 7857..7877
/label="SV40 NLS"
/note="nuclear localization signal of SV40 (simian virus
40) large T antigen"
CDS 7887..7943
/label="P2A"
/note="2A peptide from porcine teschovirus-1 polyprotein"
CDS 7944..8537
/label="PuroR"
/note="puromycin N-acetyltransferase"
misc_feature 8556..9144
/label="WPRE"
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
LTR 9216..9449
/label="3' LTR (Delta-U3)"
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
polyA_signal 9481..9705
/label="bGH poly(A) signal"
/note="bovine growth hormone polyadenylation signal"
rep_origin 9751..10179
/direction=RIGHT
/label="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(9838..9857)
/label="F1ori-R"
/note="F1 origin, reverse primer"
primer_bind 10048..10069
/label="F1ori-F"
/note="F1 origin, forward primer"
promoter 10193..10522
/label="SV40 promoter"
/note="SV40 enhancer and early promoter"
promoter 10570..10617
/label="EM7 promoter"
/note="synthetic bacterial promoter"
CDS 10636..11007
/label="BleoR"
/note="antibiotic-binding protein"
polyA_signal 11140..11273
/label="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
primer_bind complement(11310..11326)
/label="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(11310..11326)
/label="M13 Reverse"
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(11323..11345)
/label="M13/pUC Reverse"
/note="In lacZ gene"
protein_bind 11334..11350
/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(11358..11388)
/label="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind complement(11403..11424)
/label="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(11541..11558)
/label="L4440"
/note="L4440 vector, forward primer"
rep_origin complement(11712..12300)
/direction=LEFT
/label="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(12474..13331)
/label="AmpR"
/note="beta-lactamase"
promoter complement(13332..13436)
/label="AmpR promoter"
primer_bind complement(13511..13530)
/label="pRS-marker"
/note="pRS vectors, use to sequence yeast selectable
marker"
enhancer 13702..14081
/label="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"