Basic Vector Information
This is the recommended vector for cloning and expressing new shRNA sequences. This plasmid is being used by The RNAi Consortium to produce their shRNA library http://www.broad.mit.edu/genome_bio/trc/ The 1.9kb stuffer can be released with AgeI and EcoRI and replaced with your shRNA sequence of choice.
| Basic Vector Information | |||
|---|---|---|---|
| Vector Name | pLKO.1-TRC | Antibiotic Resistance | Ampicillin |
| Length | 8901 bp | Type | RNAi |
| Source | David Root | Copy Number | High copy number |
| Promoter | U6 | Cloning Method | Enzyme Cut |
| 5' Primer | LKO.15' ：GACTATCATATGCTTACCGT | Expression Method | Constiutive, Stable / Transient |
pLKO.1-TRC 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 ℃.
pLKO.1-TRC vector Sequence
LOCUS Exported 8901 bp ds-DNA circular SYN 13-DEC-2021
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS pLKO.1-TRC
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8901)
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..8901
/organism="synthetic DNA construct"
/mol_type="other DNA"
CDS 529..1128
/codon_start=1
/gene="pac from Streptomyces alboniger"
/product="puromycin N-acetyltransferase"
/note="PuroR"
/note="confers resistance to puromycin"
/translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
LTR 1256..1489
/note="3' LTR (Delta-U3)"
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
polyA_signal 1561..1682
/note="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
rep_origin 1722..1857
/note="SV40 ori"
/note="SV40 origin of replication"
promoter complement(1878..1896)
/note="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(1906..1922)
/note="M13 fwd"
/note="common sequencing primer, one of multiple similar
variants"
rep_origin 2064..2519
/direction=RIGHT
/note="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 2545..2649
/gene="bla"
/note="AmpR promoter"
CDS 2650..3510
/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 3681..4269
/direction=RIGHT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
protein_bind 4557..4578
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 4593..4623
/note="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind 4631..4647
/bound_moiety="lac repressor encoded by lacI"
/note="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 4655..4671
/note="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
promoter 4692..4710
/note="T3 promoter"
/note="promoter for bacteriophage T3 RNA polymerase"
promoter 4738..4964
/note="RSV promoter"
/note="Rous sarcoma virus enhancer/promoter"
LTR 4965..5145
/note="5' LTR (truncated)"
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 5192..5317
/note="HIV-1 Psi"
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 5810..6043
/note="RRE"
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
promoter 6570..6810
/note="U6 promoter"
/note="RNA polymerase III promoter for human U6 snRNA"
misc_feature 6822..8690
/note="stuffer"
misc_feature 8732..8849
/note="cPPT/CTS"
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 8898..507
/note="hPGK promoter"
/note="human phosphoglycerate kinase 1 promoter"
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