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pLVTHM vector (V012792#)

Basic Vector Information

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).

Basic Vector Information
Vector Name pLVTHM Antibiotic Resistance Ampicillin
Length 11085 bp Type RNAi
Source Didier Trono Copy Number High copy number
Promoter H1

pLVTHM vector Vector Map

3' LTR HIV-1 Psi RRE EF-1-alpha promoter cPPT/CTS EGFP WPRE tetracycline response element H1 promoter loxP 5' LTR (truncated) SP6 promoter bla bla ori SV40 promoter small t intron SV40 NLS SV40 poly(A) signal

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 ℃.

pLVTHM vector Sequence

Copy Sequence

Download GeneBank File(.gb)

LOCUS       Exported               11085 bp ds-DNA     circular SYN 13-DEC-2021
DEFINITION  synthetic circular DNA
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="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
     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 
     misc_feature    3347..3464
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     CDS             3554..4270
                     /product="enhanced GFP"
                     /note="mammalian codon-optimized"
     misc_feature    4343..4931
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             complement(4814..4825)
                     /product="Factor Xa recognition and cleavage site"
                     /note="Factor Xa site"
     protein_bind    5099..5369
                     /bound_moiety="tetracycline repressor TetR"
                     /note="tetracycline response element"
                     /note="contains seven copies of the tetracycline operator 
     promoter        5398..5612
                     /note="H1 promoter"
                     /note="human H1 RNA promoter"
     protein_bind    5647..5680
                     /bound_moiety="Cre recombinase"
                     /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
                     /note="AmpR promoter"
     CDS             6816..7676
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
     rep_origin      7847..8435
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
     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
                     /product="nuclear localization signal of SV40 large T 
                     /note="SV40 NLS"
     polyA_signal    10784..10918
                     /note="SV40 poly(A) signal"
                     /note="SV40 polyadenylation signal"

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