Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V000807 | pLIX_403 | 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:
- pLIX_403
- Antibiotic Resistance:
- Chloramphenicol, Ampicillin
- Length:
- 9396 bp
- Type:
- Mammalian Expression, Lentiviral ; Gateway Destina
- Replication origin:
- ori
- Selection Marker:
- Puromycin
- Promoter:
- hPGK
- Cloning Method:
- Gateway Cloning
- 5' Primer:
- LNCX
- 3' Primer:
- O.PGK1b-R (GAACGGACGTGAAGAATGTG)
pLIX_403 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.
pLIX_403 vector Sequence
LOCUS 40924_28312 9396 bp DNA circular SYN 13-MAY-2021
DEFINITION Inducible lentiviral expression, TRE-gateway-V5; PGK-puro-2A-rtTA.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 9396)
TITLE Root Lab pLEX and pLIX Gateway backbones
JOURNAL Unpublished
REFERENCE 2 (bases 1 to 9396)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 9396)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName:
"Unpublished"
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..9396
/mol_type="other DNA"
/organism="synthetic DNA construct"
misc_feature 41..158
/label=cPPT/CTS
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 212..526
/label=tight TRE promoter
/note="Tet-responsive promoter PTight, consisting of seven
tet operator sequences followed by the minimal CMV
promoter"
protein_bind 547..671
/label=attR1
/note="recombination site for the Gateway(R) LR reaction"
promoter 696..726
/label=lac UV5 promoter
/note="E. coli lac promoter with an 'up' mutation"
CDS 780..1436
/codon_start=1
/label=CmR
/note="chloramphenicol acetyltransferase"
/translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
CDS 1781..2083
/codon_start=1
/label=ccdB
/note="CcdB, a bacterial toxin that poisons DNA gyrase"
/translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
protein_bind complement(2127..2251)
/label=attR2
/note="recombination site for the Gateway(R) LR reaction"
CDS 2256..2297
/codon_start=1
/label=V5 tag
/note="epitope tag from simian virus 5"
/translation="GKPIPNPLLGLDST"
CDS 2311..2328
/codon_start=1
/label=6xHis
/note="6xHis affinity tag"
/translation="HHHHHH"
promoter 2338..2848
/label=hPGK promoter
/note="human phosphoglycerate kinase 1 promoter"
CDS 2858..3454
/codon_start=1
/label=PuroR
/note="puromycin N-acetyltransferase"
/translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
CDS 3455..3508
/codon_start=1
/label=T2A
/note="2A peptide from Thosea asigna virus capsid protein"
/translation="EGRGSLLTCGDVEENPGP"
CDS 3509..4252
/codon_start=1
/label=rtTA-Advanced
/note="improved tetracycline-controlled transactivator"
/translation="MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
KNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
DSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
DALDDFDLDMLPADALDDFDLDMLPG"
primer_bind complement(4295..4315)
/label=WPRE-R
/note="WPRE, reverse primer"
CDS complement(4713..4724)
/codon_start=1
/label=Factor Xa site
/note="Factor Xa recognition and cleavage site"
/translation="IEGR"
LTR 4929..5162
/label=3' LTR (Delta-U3)
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
polyA_signal 5234..5368
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
rep_origin 5395..5530
/label=SV40 ori
/note="SV40 origin of replication"
promoter 5531..5601
/label=AmpR promoter
CDS 5602..6459
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 6633..7221
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 7375..7392
/label=L4440
/note="L4440 vector, forward primer"
protein_bind 7509..7530
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 7545..7575
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 7583..7599
/label=lac operator
/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)."
primer_bind 7607..7623
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
promoter 7644..7662
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
promoter 7690..7916
/label=RSV promoter
/note="Rous sarcoma virus enhancer/promoter"
LTR 7917..8097
/label=5' LTR (truncated)
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 8144..8269
/label=HIV-1 Psi
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 8762..8995
/label=RRE
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 9180..9224
/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"