CRISPRi library backbone 载体 (V012348#)

载体基本信息
载体名称 CRISPRi library backbone 载体抗性 Ampicillin
载体长度 8888 bp 载体类型 CRISPR Plasmids
载体来源 Gilbert LA, Horlbeck MA, Adamson B, Villalta JE, Chen Y, Whitehead 拷贝数 High copy number

CRISPRi library backbone 载体载体图谱

CRISPRi library backbone8888 bp40080012001600200024002800320036004000440048005200560060006400680072007600800084008800CMV enhancerCMV promoterLTR (truncated)HIV-1 PsiRREgp41 peptideProtein Tat from Human immunodeficiency virus type 1 group M subtype B (isolate WMJ22). Accession#: P12509cPPT/CTSU6 promotergRNA scaffoldpolIII terminatorEF-1-alpha promoterPuroRT2ATagBFPloxPWPREKS primer5' LTR (truncated)oriAmpRAmpR promoter

CRISPRi library backbone 载体载体序列

LOCUS       CRISPRi_library        8888 bp DNA     circular SYN 01-JAN-1980
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
REFERENCE   3  (bases 1 to 8888)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Cell"; 
            date: "2014"; volume: "159"; pages: "647-61"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..8888
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        247..626
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        627..829
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     LTR             844..1024
                     /label=LTR (truncated)
                     /note="LTR (truncated)"
                     /note="truncated long terminal repeat (LTR) from HIV-1"
     misc_feature    1071..1196
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus
                     type 1"
     misc_feature    1695..1928
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2113..2157
                     /label=gp41 peptide
                     /note="antigenic peptide corresponding to amino acids 655
                     to 669 of the HIV envelope protein gp41 (Lutje Hulsik et 
                     al., 2013)"
     CDS             2306..2347
                     /note="Protein Tat from Human immunodeficiency virus type 1
                     group M subtype B (isolate WMJ22). Accession#: P12509"
     misc_feature    2455..2572
                     /label=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
                     /label=Cre recombinase binding site
                     /bound_moiety="Cre recombinase"
                     /note="loxP"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     misc_feature    2965..3050
                     /label=gRNA scaffold
                     /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        3114..4286
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation
                     factor EF-1-alpha"
     CDS             4298..4894
                     /label=PuroR
                     /note="puromycin N-acetyltransferase"
     CDS             4904..4957
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid
                     protein"
                     /label=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."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             4970..5665
                     /label=TagBFP
                     /note="monomeric blue fluorescent protein"
     protein_bind    complement(5688..5721)
                     /label=loxP
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    5777..6365
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional
                     regulatory element"
     primer_bind     complement(6368..6384)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     LTR             6894..7074
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(7136..7724)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(7898..8755)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(8756..8860)
                     /label=AmpR promoter

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