LOCUS       V012348                 8888 bp    DNA     circular SYN 01-JAN-1980
DEFINITION  Exported.
ACCESSION   V012348
VERSION     V012348
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"
            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"
                     /label="Protein Tat"
     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"
ORIGIN
        1 gcttaagcgg tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa
       61 tctgctctga tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg
      121 ctgagtagtg cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca
      181 tgaagaatct gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata
      241 cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc
      301 atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac
      361 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa
      421 tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag
      481 tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc
      541 ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct
      601 acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg
      661 gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt
      721 tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga
      781 cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagcgcg ttttgcctgt
      841 actgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac
      901 ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg
      961 ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct
     1021 agcagtggcg cccgaacagg gacttgaaag cgaaagggaa accagaggag ctctctcgac
     1081 gcaggactcg gcttgctgaa gcgcgcacgg caagaggcga ggggcggcga ctggtgagta
     1141 cgccaaaaat tttgactagc ggaggctaga aggagagaga tgggtgcgag agcgtcagta
     1201 ttaagcgggg gagaattaga tcgcgatggg aaaaaattcg gttaaggcca gggggaaaga
     1261 aaaaatataa attaaaacat atagtatggg caagcaggga gctagaacga ttcgcagtta
     1321 atcctggcct gttagaaaca tcagaaggct gtagacaaat actgggacag ctacaaccat
     1381 cccttcagac aggatcagaa gaacttagat cattatataa tacagtagca accctctatt
     1441 gtgtgcatca aaggatagag ataaaagaca ccaaggaagc tttagacaag atagaggaag
     1501 agcaaaacaa aagtaagacc accgcacagc aagcggccgg ccgcgctgat cttcagacct
     1561 ggaggaggag atatgaggga caattggaga agtgaattat ataaatataa agtagtaaaa
     1621 attgaaccat taggagtagc acccaccaag gcaaagagaa gagtggtgca gagagaaaaa
     1681 agagcagtgg gaataggagc tttgttcctt gggttcttgg gagcagcagg aagcactatg
     1741 ggcgcagcgt caatgacgct gacggtacag gccagacaat tattgtctgg tatagtgcag
     1801 cagcagaaca atttgctgag ggctattgag gcgcaacagc atctgttgca actcacagtc
     1861 tggggcatca agcagctcca ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa
     1921 cagctcctgg ggatttgggg ttgctctgga aaactcattt gcaccactgc tgtgccttgg
     1981 aatgctagtt ggagtaataa atctctggaa cagatttgga atcacacgac ctggatggag
     2041 tgggacagag aaattaacaa ttacacaagc ttaatacact ccttaattga agaatcgcaa
     2101 aaccagcaag aaaagaatga acaagaatta ttggaattag ataaatgggc aagtttgtgg
     2161 aattggttta acataacaaa ttggctgtgg tatataaaat tattcataat gatagtagga
     2221 ggcttggtag gtttaagaat agtttttgct gtactttcta tagtgaatag agttaggcag
     2281 ggatattcac cattatcgtt tcagacccac ctcccaaccc cgaggggacc cgacaggccc
     2341 gaaggaatag aagaagaagg tggagagaga gacagagaca gatccattcg attagtgaac
     2401 ggatcggcac tgcgtgcgcc aattctgcag acaaatggca gtattcatcc acaattttaa
     2461 aagaaaaggg gggattgggg ggtacagtgc aggggaaaga atagtagaca taatagcaac
     2521 agacatacaa actaaagaat tacaaaaaca aattacaaaa attcaaaatt ttcgggttta
     2581 ttacagggac agcagagatc cagtttggtt agtaccgggc ccgctctaga gatccgacgc
     2641 gccatctcta ggcccgcgcc ggccccctcg cacggacttg tgggagaagc tcggctactc
     2701 ccctgccccg gttaatttgc atataatatt tcctagtaac tatagaggct taatgtgcga
     2761 taaaagacag ataatctgtt ctttttaata ctagctacat tttacatgat aggcttggat
     2821 ttctataact tcgtatagca tacattatac gaagttataa acagcacaaa aggaaactca
     2881 ccctaactgt aaagtaattg tgtgttttga gactataagt atcccttgga gaaccacctt
     2941 gttggaccag gatgggcacc acccgtttaa gagctaagct ggaaacagca tagcaagttt
     3001 aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc tttttttctc
     3061 gagtactagg atccattagg cggccgcgtg gataaccgta ttaccgccat gcatgtgccc
     3121 gtcagtgggc agagcgcaca tcgcccacag tccccgagaa gttgggggga ggggtcggca
     3181 attgaaccgg tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact
     3241 ggctccgcct ttttcccgag ggtgggggag aaccgtatat aagtgcagta gtcgccgtga
     3301 acgttctttt tcgcaacggg tttgccgcca gaacacaggt aagtgccgtg tgtggttccc
     3361 gcgggcctgg cctctttacg ggttatggcc cttgcgtgcc ttgaattact tccacctggc
     3421 tgcagtacgt gattcttgat cccgagcttc gggttggaag tgggtgggag agttcgaggc
     3481 cttgcgctta aggagcccct tcgcctcgtg cttgagttga ggcctggcct gggcgctggg
     3541 gccgccgcgt gcgaatctgg tggcaccttc gcgcctgtct cgctgctttc gataagtctc
     3601 tagccattta aaatttttga tgacctgctg cgacgctttt tttctggcaa gatagtcttg
     3661 taaatgcggg ccaagatctg cacactggta tttcggtttt tggggccgcg ggcggcgacg
     3721 gggcccgtgc gtcccagcgc acatgttcgg cgaggcgggg cctgcgagcg cggccaccga
     3781 gaatcggacg ggggtagtct caagctggcc ggcctgctct ggtgcctggc ctcgcgccgc
     3841 cgtgtatcgc cccgccctgg gcggcaaggc tggcccggtc ggcaccagtt gcgtgagcgg
     3901 aaagatggcc gcttcccggc cctgctgcag ggagctcaaa atggaggacg cggcgctcgg
     3961 gagagcgggc gggtgagtca cccacacaaa ggaaaagggc ctttccgtcc tcagccgtcg
     4021 cttcatgtga ctccacggag taccgggcgc cgtccaggca cctcgattag ttctcgcgct
     4081 tttggagtac gtcgtcttta ggttgggggg aggggtttta tgcgatggag tttccccaca
     4141 ctgagtgggt ggagactgaa gttaggccag cttggcactt gatgtaattc tccttggaat
     4201 ttgccctttt tgagtttgga tcttggttca ttctcaagcc tcagacagtg gttcaaagtt
     4261 tttttcttcc atttcaggtg tcgtgagcta gcccaccatg accgagtaca agcccacggt
     4321 gcgcctcgcc acccgcgacg acgtcccccg ggccgtacgc accctcgccg ccgcgttcgc
     4381 cgactacccc gccacgcgcc acaccgtcga cccggaccgc cacatcgagc gggtcaccga
     4441 gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga
     4501 cgacggcgcc gcggtggcgg tctggaccac gccggagagc gtcgaagcgg gggcggtgtt
     4561 cgccgagatc ggcccgcgca tggccgagtt gagcggttcc cggctggccg cgcagcaaca
     4621 gatggaaggc ctcctggcgc cgcaccggcc caaggagccc gcgtggttcc tggccaccgt
     4681 cggcgtctcg cccgaccacc agggcaaggg tctgggcagc gccgtcgtgc tccccggagt
     4741 ggaggcggcc gagcgcgccg gggtgcccgc cttcctggag acctccgcgc cccgcaacct
     4801 ccccttctac gagcggctcg gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc
     4861 gcgcacctgg tgcatgaccc gcaagcccgg tgccggatcg ggagagggca gaggaagtct
     4921 gctaacatgc ggtgacgtcg aggagaatcc tggcccaccg gtcgccacca gcgagctgat
     4981 taaggagaac atgcacatga agctgtacat ggagggcacc gtggacaacc atcacttcaa
     5041 gtgcacatcc gagggcgaag gcaagcccta cgagggcacc cagaccatga gaatcaaggt
     5101 ggtcgagggc ggccctctcc ccttcgcctt cgacatcctg gctactagct tcctctacgg
     5161 cagcaagacc ttcatcaacc acacccaggg catccccgac ttcttcaagc agtccttccc
     5221 tgagggcttc acatgggaga gagtcaccac atacgaagac gggggcgtgc tgaccgctac
     5281 ccaggacacc agcctccagg acggctgcct catctacaac gtcaagatca gaggggtgaa
     5341 cttcacatcc aacggccctg tgatgcagaa gaaaacactc ggctgggagg ccttcaccga
     5401 gacgctgtac cccgctgacg gcggcctgga aggcagaaac gacatggccc tgaagctcgt
     5461 gggcgggagc catctgatcg caaacatcaa gaccacatat agatccaaga aacccgctaa
     5521 gaacctcaag atgcctggcg tctactatgt ggactacaga ctggaaagaa tcaaggaggc
     5581 caacaacgag acctacgtcg agcagcacga ggtggcagtg gccagatact gcgacctccc
     5641 tagcaaactg gggcacaagc ttaattaaga attcgtcgag ggacctaata acttcgtata
     5701 gcatacatta tacgaagtta tacatgttta agggttccgg ttccactagg tacaattcga
     5761 tatcaagctt atcgataatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat
     5821 tcttaactat gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca
     5881 tgctattgct tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc
     5941 tctttatgag gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc
     6001 tgacgcaacc cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt
     6061 cgctttcccc ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg
     6121 gacaggggct cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc
     6181 ctttccttgg ctgctcgcct gtgttgccac ctggattctg cgcgggacgt ccttctgcta
     6241 cgtcccttcg gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg
     6301 gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc
     6361 cccgcatcga taccgtcgac ctcgatcgag acctagaaaa acatggagca atcacaagta
     6421 gcaatacagc agctaccaat gctgattgtg cctggctaga agcacaagag gaggaggagg
     6481 tgggttttcc agtcacacct caggtacctt taagaccaat gacttacaag gcagctgtag
     6541 atcttagcca ctttttaaaa gaaaaggggg gactggaagg gctaattcac tcccaacgaa
     6601 gacaagatat ccttgatctg tggatctacc acacacaagg ctacttccct gattggcaga
     6661 actacacacc agggccaggg atcagatatc cactgacctt tggatggtgc tacaagctag
     6721 taccagttga gcaagagaag gtagaagaag ccaatgaagg agagaacacc cgcttgttac
     6781 accctgtgag cctgcatggg atggatgacc cggagagaga agtattagag tggaggtttg
     6841 acagccgcct agcatttcat cacatggccc gagagctgca tccggactgt actgggtctc
     6901 tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta
     6961 agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact
     7021 ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagcatgt
     7081 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     7141 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     7201 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     7261 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     7321 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     7381 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     7441 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     7501 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     7561 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     7621 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     7681 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     7741 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     7801 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     7861 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     7921 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     7981 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     8041 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     8101 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     8161 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     8221 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     8281 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     8341 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     8401 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     8461 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     8521 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     8581 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     8641 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     8701 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     8761 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     8821 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     8881 cacctgac
//
