Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V016843 | pCW-eGFP-SHLD2 | 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:
- pCW-eGFP-SHLD2
- Antibiotic Resistance:
- Ampicillin
- Length:
- 11155 bp
- Type:
- Protein expression
- Replication origin:
- ori
- Host:
- Mammalian cells, Lentivirus
- Selection Marker:
- Puro
- Promoter:
- hPGK
- Growth Strain(s):
- Stbl3
- Growth Temperature:
- 30℃
pCW-eGFP-SHLD2 vector 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.
pCW-eGFP-SHLD2 vector Sequence
LOCUS 62056_8135 11155 bp DNA circular SYN 01-JAN-1980
DEFINITION synthetic circular DNA.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 11155)
AUTHORS .
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..11155
/mol_type="other DNA"
/organism="synthetic DNA construct"
misc_feature 43..160
/label=cPPT/CTS
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 214..528
/label=tight TRE promoter
/note="Tet-responsive promoter PTight, consisting of seven
tet operator sequences followed by the minimal CMV
promoter"
protein_bind 549..573
/label=attB1
/note="recombination site for the Gateway(R) BP reaction"
CDS 591..1307
/codon_start=1
/label=EGFP
/note="enhanced GFP"
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
EFVTAAGITLGMDELYK"
protein_bind complement(4030..4054)
/label=attB2
/note="recombination site for the Gateway(R) BP reaction"
CDS 4072..4089
/codon_start=1
/label=6xHis
/note="6xHis affinity tag"
/translation="HHHHHH"
promoter 4099..4609
/label=hPGK promoter
/note="human phosphoglycerate kinase 1 promoter"
CDS 4619..5215
/codon_start=1
/label=PuroR
/note="puromycin N-acetyltransferase"
/translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
CDS 5216..5269
/codon_start=1
/label=T2A
/note="2A peptide from Thosea asigna virus capsid protein"
/translation="EGRGSLLTCGDVEENPGP"
CDS 5270..6013
/codon_start=1
/label=rtTA-Advanced
/note="improved tetracycline-controlled transactivator"
/translation="MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
KNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
DSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
DALDDFDLDMLPADALDDFDLDMLPG"
CDS complement(6474..6485)
/codon_start=1
/label=Factor Xa site
/note="Factor Xa recognition and cleavage site"
/translation="IEGR"
LTR 6690..6923
/label=3' LTR (Delta-U3)
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
polyA_signal 6995..7129
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
rep_origin 7156..7291
/label=SV40 ori
/note="SV40 origin of replication"
promoter 7292..7362
/label=AmpR promoter
CDS 7363..8220
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 8394..8982
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
protein_bind 9270..9291
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 9306..9336
/label=lac promoter
/note="promoter for the E. coli lac operon"
promoter 9405..9423
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
promoter 9451..9677
/label=RSV promoter
/note="Rous sarcoma virus enhancer/promoter"
misc_feature 10523..10756
/label=RRE
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 10941..10985
/codon_start=1
/label=gp41 peptide
/note="antigenic peptide corresponding to amino acids 655
to 669 of the HIV envelope protein gp41 (Lutje Hulsik et
al., 2013)"
/translation="KNEQELLELDKWASL"