CRISPRi library backbone vector (V012348#)

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

CRISPRi library backbone8888 bp40080012001600200024002800320036004000440048005200560060006400680072007600800084008800CMV enhancerCMV promoterLTR (truncated)HIV-1 PsiRREcPPT/CTSU6 promotergRNA scaffoldpolIII terminatorEF-1-alpha core promoterEF-1-alpha intron APuroRT2ATagBFPloxPWPRELTR (truncated)oriAmpRAmpR promoter

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

Copy Sequence

Download GeneBank File(.gb)

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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