pLVTHM allows for direct cloning of shRNAs into the lentiviral vectors and replaces the old pLVTH system from Trono lab. pLVTHM is similar to pLVTH, but contains a 3bp substitution that generates a unique MluI site for direct cloning of an shRNA into MluI-ClaI. Please note that there is an additional ClaI site in this vector that is blocked by Dam methylation (this site does not appear in the depositor's full sequence). The plasmid needs to be grown in a Dam+ bacterial strain in order to use ClaI for cloning. Note that ClaI has lower salt concentration requirements than MluI. One way to handle this is to digest with ClaI for 45 minutes, followed by addition of diluted MluI buffer and MluI, then incubation for 90 more minutes. Be sure not to use a large amount of vector (1.7 ug to 2.5 ug is known to work). Also, if your shRNA is already in pSUPER (or another plasmid under the control of the PolIII promoter) you may use the EcoRI-ClaI sites to replace the H1 promoter in pLVTHM with the H1-shRNA cassette from pSUPER (or another plasmid).
| 载体基本信息 | |||
| 载体名称 | pLVTHM | 载体抗性 | Ampicillin |
|---|---|---|---|
| 载体长度 | 11085 bp | 载体类型 | RNAi |
| 载体来源 | Didier Trono | 拷贝数 | High copy number |
| 启动子 | H1 | ||
pLVTHM 载体载体图谱
pLVTHM 载体载体序列
LOCUS Exported 11085 bp ds-DNA circular SYN 13-DEC-2021
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS pLVTHM
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 11085)
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..11085
/organism="synthetic DNA construct"
/mol_type="other DNA"
LTR 2..635
/note="3 LTR"
/note="3 long terminal repeat (LTR) from HIV-1"
misc_feature 682..807
/note="HIV-1 Psi"
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 1300..1533
/note="RRE"
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
promoter 2122..3299
/note="EF-1-alpha promoter"
/note="strong constitutive promoter for human elongation
factor EF-1-alpha"
intron 2353..3290
/note="EF-1-alpha intron A"
/note="intron upstream of the start codon of human
EF-1-alpha"
misc_feature 3347..3464
/note="cPPT/CTS"
/note="central polypurine tract and central termination
sequence of HIV-1"
CDS 3554..4270
/codon_start=1
/product="enhanced GFP"
/note="EGFP"
/note="mammalian codon-optimized"
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
EFVTAAGITLGMDELYK"
misc_feature 4343..4931
/note="WPRE"
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
CDS complement(4814..4825)
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/note="Factor Xa site"
/translation="IEGR"
protein_bind 5099..5369
/bound_moiety="tetracycline repressor TetR"
/note="tetracycline response element"
/note="contains seven copies of the tetracycline operator
tetO"
promoter 5398..5612
/note="H1 promoter"
/note="human H1 RNA promoter"
protein_bind 5647..5680
/bound_moiety="Cre recombinase"
/note="loxP"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT)."
LTR 5700..5880
/note="5 LTR (truncated)"
/note="truncated 5 long terminal repeat (LTR) from HIV-1"
promoter complement(5905..5923)
/note="SP6 promoter"
/note="promoter for bacteriophage SP6 RNA polymerase"
promoter 6711..6815
/gene="bla"
/note="AmpR promoter"
CDS 6816..7676
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/note="AmpR"
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 7847..8435
/direction=RIGHT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
promoter 8681..9010
/note="SV40 promoter"
/note="SV40 enhancer and early promoter"
rep_origin 8861..8996
/note="SV40 ori"
/note="SV40 origin of replication"
intron 10144..10209
/note="small t intron"
/note="simian virus 40 (SV40) small t antigen intron"
CDS 10339..10359
/codon_start=1
/product="nuclear localization signal of SV40 large T
antigen"
/note="SV40 NLS"
/translation="PKKKRKV"
polyA_signal 10784..10918
/note="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
