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"