Basic Vector Information
- Vector Name:
- pLVCT-tTR-KRAB
- Antibiotic Resistance:
- Ampicillin
- Length:
- 12884 bp
- Type:
- Mammalian Expression, Lentiviral
- Replication origin:
- ori
- Copy Number:
- High Copy
- Promoter:
- CAG
- Cloning Method:
- Restriction Enzyme
- 5' Primer:
- See map
pLVCT-tTR-KRAB vector Vector Map
Plasmid Resuspension 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 ℃.
pLVCT-tTR-KRAB vector Sequence
LOCUS Exported 12884 bp ds-DNA circular SYN 13-MAY-2021
DEFINITION Tet-regulated (Tet-on) lentiviral vector for transgene (CAG
promoter) - OR - shRNA (H1 promoter when subcloned from pLVTHM
(Addgene#12247)) - 2nd generation.
ACCESSION .
VERSION .
KEYWORDS pLVCT-tTR-KRAB
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 12884)
AUTHORS Szulc J, Wiznerowicz M, Sauvain MO, Trono D, Aebischer P
TITLE A versatile tool for conditional gene expression and knockdown.
JOURNAL Nat Methods. 2006 Feb . 3(2):109-16.
PUBMED 16432520
REFERENCE 2 (bases 1 to 12884)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Nat
Methods. 2006 Feb . 3(2):109-16."
FEATURES Location/Qualifiers
source 1..12884
/organism="synthetic DNA construct"
/mol_type="other DNA"
LTR 6..639
/label=3' LTR
/note="3' long terminal repeat (LTR) from HIV-1"
misc_feature 686..811
/label=HIV-1 Psi
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 1304..1537
/label=RRE
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 1722..1766
/codon_start=1
/product="antigenic peptide corresponding to amino acids
655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik
et al., 2013)"
/label=gp41 peptide
/note="recognized by the 2H10 single-chain llama nanobody"
/translation="KNEQELLELDKWASL"
enhancer 2061..2440
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 2442..2719
/label=chicken beta-actin promoter
intron 2720..3736
/label=chimeric intron
/note="chimera between introns from chicken beta-actin and
rabbit beta-globin"
primer_bind 3660..3682
/label=pCAGGS-5
/note="Chimeric intron in CAG promoter, forward primer"
primer_bind 3744..3763
/label=pCAG-F
/note="Rabbit beta-globin intron, for pCAG plasmids,
forward primer"
misc_feature 3822..3939
/label=cPPT/CTS
/note="central polypurine tract and central termination
sequence of HIV-1"
regulatory 4016..4025
/regulatory_class="other"
/label=Kozak sequence
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 4022..4738
/codon_start=1
/product="the original enhanced GFP (Yang et al., 1996)"
/label=EGFP
/note="mammalian codon-optimized"
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
EFVTAAGITLGMDELYK"
CDS 4022..4738
/codon_start=1
/product="enhanced GFP"
/label=EGFP
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
EFVTAAGITLGMDELYK"
primer_bind complement(4067..4088)
/label=EGFP-N
/note="EGFP, reverse primer"
primer_bind complement(4328..4347)
/label=EXFP-R
/note="For distinguishing EGFP variants, reverse primer"
primer_bind 4675..4696
/label=EGFP-C
/note="EGFP, forward primer"
misc_feature 4834..5385
/label=IRES
/note="internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)"
primer_bind complement(4978..4995)
/label=IRES reverse
/note="IRES internal ribosome entry site, reverse primer.
Also called pCDH-rev"
primer_bind 5205..5224
/label=IRES-F
/note="IRES internal ribosome entry site, forward primer"
CDS 5388..6005
/codon_start=1
/gene="tetR from transposon Tn10"
/product="tetracycline repressor TetR"
/label=TetR
/note="TetR binds to the tetracycline operator tetO to
inhibit transcription. This inhibition can be relieved by
adding tetracycline or doxycycline."
/translation="MARLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESG"
primer_bind complement(5461..5480)
/label=Tet-R
/note="Tetracycline resistance gene, reverse primer"
CDS 6009..6029
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 6132..6326
/codon_start=1
/product="Kruppel-associated box (KRAB) transcriptional
repression domain from the human zinc finger protein ZNF10
(Margolin et al., 1994)"
/label=KRAB
/translation="RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGY
QLTKPDVILRLEKGEEPWLV"
misc_feature 6469..7057
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
primer_bind complement(6522..6542)
/label=WPRE-R
/note="WPRE, reverse primer"
CDS complement(6940..6951)
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/label=Factor Xa site
/translation="IEGR"
protein_bind 7195..7465
/label=tetracycline response element
/bound_moiety="tetracycline repressor TetR"
/note="contains seven copies of the tetracycline operator
tetO"
LTR 7504..7684
/label=5' LTR (truncated)
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
promoter complement(7709..7727)
/label=SP6 promoter
/note="promoter for bacteriophage SP6 RNA polymerase"
primer_bind complement(7710..7727)
/label=SP6
/note="SP6 promoter, forward primer"
primer_bind complement(8017..8036)
/label=pBRrevBam
/note="pBR322 vectors, tet region, downstream of BamHI,
reverse primer"
primer_bind complement(8250..8269)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 8369..8391
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind complement(8429..8447)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 8515..8619
/gene="bla"
/label=AmpR promoter
CDS 8620..9480
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
primer_bind complement(8838..8857)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 9651..10239
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 10140..10159
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 10393..10410
/label=L4440
/note="L4440 vector, forward primer"
primer_bind complement(10480..10500)
/label=pBABE 3'
/note="SV40 enhancer, reverse primer for pBABE vectors"
promoter 10485..10814
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
rep_origin 10665..10800
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind 10727..10746
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
intron 11948..12013
/label=small t intron
/note="SV40 (simian virus 40) small t antigen intron"
CDS 12143..12163
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
polyA_signal 12588..12722
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(12625..12644)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 12679..12698
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
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