Basic Vector Information
| Basic Vector Information | |||
|---|---|---|---|
| Vector Name | CRISPRi library backbone | Antibiotic Resistance | Ampicillin |
| Length | 8888 bp | Type | CRISPR Plasmids |
| Source | Gilbert LA, Horlbeck MA, Adamson B, Villalta JE, Chen Y, Whitehead | Copy Number | High copy number |
CRISPRi library backbone 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 ℃.
CRISPRi library backbone vector Sequence
LOCUS Exported 8888 bp ds-DNA circular SYN 12-1-2016
DEFINITION Backbone vector for generating a CRISPR-mediated transcriptional
repression (CRISPRi) library.
ACCESSION .
VERSION .
KEYWORDS CRISPRi library backbone
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8888)
AUTHORS Gilbert LA, Horlbeck MA, Adamson B, Villalta JE, Chen Y, Whitehead
EH, Guimaraes C, Panning B, Ploegh HL, Bassik MC, Qi LS, Kampmann M,
Weissman JS.
TITLE Genome-Scale CRISPR-Mediated Control of Gene Repression and
Activation.
JOURNAL Cell 2014;159:647-61.
PUBMED 25307932
REFERENCE 2 (bases 1 to 8888)
AUTHORS Weissman Lab / Addgene #62217
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..8888
/organism="synthetic DNA construct"
/lab_host="Mammalian Cells"
/mol_type="other DNA"
enhancer 247..626
/note="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"
promoter 628..826
/note="CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
LTR 844..1024
/note="LTR (truncated)"
/note="truncated long terminal repeat (LTR) from HIV-1"
misc_feature 1071..1196
/note="HIV-1 Psi"
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 1695..1928
/note="RRE"
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
misc_feature 2455..2572
/note="cPPT/CTS"
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 2631..2943
/note="U6 promoter"
/note="modified RNA polymerase III promoter for mouse U6
snRNA"
protein_bind 2825..2858
/bound_moiety="Cre recombinase"
/note="loxP"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT)."
misc_feature 2965..3050
/note="gRNA scaffold"
/note="modified guide RNA scaffold for the Streptococcus
pyogenes CRISPR/Cas9 system"
terminator 3051..3056
/note="polIII terminator"
/note="RNA polymerase III transcription terminator"
promoter 3127..3338
/note="EF-1-alpha core promoter"
/note="core promoter for human elongation factor
EF-1-alpha"
intron 3339..4277
/note="EF-1-alpha intron A"
/note="intron upstream of the start codon of human
EF-1-alpha"
CDS 4298..4894
/codon_start=1
/gene="pac from Streptomyces alboniger"
/product="puromycin N-acetyltransferase"
/note="PuroR"
/note="confers resistance to puromycin"
/translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
CDS 4904..4957
/codon_start=1
/product="2A peptide from Thosea asigna virus capsid
protein"
/note="T2A"
/note="Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides."
/protein_id="
"
/translation="EGRGSLLTCGDVEENPGP"
CDS 4970..5668
/codon_start=1
/product="monomeric blue fluorescent protein"
/note="TagBFP"
/note="mammalian codon-optimized"
/translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
LPSKLGHKLN"
protein_bind 5688..5721
/bound_moiety="Cre recombinase"
/note="loxP"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT)."
misc_feature 5777..6365
/note="WPRE"
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
LTR 6894..7074
/note="LTR (truncated)"
/note="truncated long terminal repeat (LTR) from HIV-1"
rep_origin complement(7136..7724)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(7895..8755)
/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"
promoter complement(8756..8860)
/gene="bla"
/note="AmpR promoter"
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