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
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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