LOCUS       lenti_sgRNA(MS2)        9996 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Zhang lab lentiviral vector for expressing a Zeocin(TM)-resistance 
            marker plus a single guide RNA (sgRNA) containing two MS2 stem 
            loops.
ACCESSION   .
VERSION     .
KEYWORDS    lenti sgRNA(MS2)_zeo backbone.
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9996)
  AUTHORS   Konermann S, Brigham MD, Trevino AE, Joung J, Abudayyeh OO, Barcena 
            C, Hsu PD, Habib N, Gootenberg JS, Nishimasu H, Nureki O, Zhang F.
  TITLE     Genome-scale transcriptional activation by an engineered CRISPR-Cas9
            complex.
  JOURNAL   Nature 2015;517:583-8.
  PUBMED    25494202
REFERENCE   2  (bases 1 to 9996)
  AUTHORS   Zhang Lab / Addgene #61427
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 9996)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nature"; 
            date: "2015"; volume: "517"; pages: "583-8"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..9996
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        238..617
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        618..820
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     LTR             835..1015
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1062..1187
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus
                     type 1"
     misc_feature    1680..1913
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2098..2142
                     /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             2291..2332
                     /note="Protein Tat from Human immunodeficiency virus type 1
                     group M subtype B (isolate WMJ22). Accession#: P12509"
     misc_feature    2440..2557
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination
                     sequence of HIV-1"
     promoter        2607..2847
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_RNA        2893..2911
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat
                     protein"
     misc_RNA        2963..2981
                     /label=MS2 stem loop
                     /note="MS2 stem loop"
                     /note="stem loop that binds the bacteriophage MS2 coat
                     protein"
     terminator      3012..3017
                     /note="polIII terminator"
                     /note="RNA polymerase III transcription terminator"
     promoter        3105..4282
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation
                     factor EF-1-alpha"
     CDS             4289..4660
                     /label=BleoR
                     /note="antibiotic-binding protein"
     misc_feature    4688..5276
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional
                     regulatory element"
     LTR             5801..5981
                     /label=5' LTR (truncated)
                     /note="5' LTR (truncated)"
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     polyA_signal    6013..6237
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      6283..6711
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        6725..7054
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     promoter        7102..7149
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter"
     CDS             7168..7539
                     /label=BleoR
                     /note="antibiotic-binding protein"
     polyA_signal    7672..7805
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        complement(7890..7920)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(7935..7956)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(8244..8832)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(9006..9863)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(9864..9968)
                     /label=AmpR promoter
ORIGIN
        1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
       61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
      121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
      181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
      241 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
      301 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
      361 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
      421 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
      481 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      541 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
      601 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
      661 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
      721 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
      781 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct
      841 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
      901 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
      961 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc
     1021 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc
     1081 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa
     1141 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg
     1201 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata
     1261 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc
     1321 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga
     1381 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc
     1441 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca
     1501 aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg
     1561 agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga
     1621 gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata
     1681 ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg
     1741 acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg
     1801 ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag
     1861 ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt
     1921 tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt
     1981 aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt
     2041 aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag
     2101 aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata
     2161 acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta
     2221 agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta
     2281 tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa
     2341 gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc ggcactgcgt
     2401 gcgccaattc tgcagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat
     2461 tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa
     2521 agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag
     2581 agatccagtt tggttaatta gctagcgagg gcctatttcc catgattcct tcatatttgc
     2641 atatacgata caaggctgtt agagagataa ttggaattaa tttgactgta aacacaaaga
     2701 tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt gcagttttaa
     2761 aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct
     2821 tggctttata tatcttgtgg aaaggacgaa acaccggaga cgggataccg tctctgtttt
     2881 agagctaggc caacatgagg atcacccatg tctgcagggc ctagcaagtt aaaataaggc
     2941 tagtccgtta tcaacttggc caacatgagg atcacccatg tctgcagggc caagtggcac
     3001 cgagtcggtg ctttttttgg atcctgcaaa gatggataaa gttttaaaca gagaggaatc
     3061 tttgcagcta atggaccttc taggtcttga aaggagtggg aattggctcc ggtgcccgtc
     3121 agtgggcaga gcgcacatcg cccacagtcc ccgagaagtt ggggggaggg gtcggcaatt
     3181 gatccggtgc ctagagaagg tggcgcgggg taaactggga aagtgatgtc gtgtactggc
     3241 tccgcctttt tcccgagggt gggggagaac cgtatataag tgcagtagtc gccgtgaacg
     3301 ttctttttcg caacgggttt gccgccagaa cacaggtaag tgccgtgtgt ggttcccgcg
     3361 ggcctggcct ctttacgggt tatggccctt gcgtgccttg aattacttcc actggctgca
     3421 gtacgtgatt cttgatcccg agcttcgggt tggaagtggg tgggagagtt cgaggccttg
     3481 cgcttaagga gccccttcgc ctcgtgcttg agttgaggcc tggcctgggc gctggggccg
     3541 ccgcgtgcga atctggtggc accttcgcgc ctgtctcgct gctttcgata agtctctagc
     3601 catttaaaat ttttgatgac ctgctgcgac gctttttttc tggcaagata gtcttgtaaa
     3661 tgcgggccaa gatctgcaca ctggtatttc ggtttttggg gccgcgggcg gcgacggggc
     3721 ccgtgcgtcc cagcgcacat gttcggcgag gcggggcctg cgagcgcggc caccgagaat
     3781 cggacggggg tagtctcaag ctggccggcc tgctctggtg cctggcctcg cgccgccgtg
     3841 tatcgccccg ccctgggcgg caaggctggc ccggtcggca ccagttgcgt gagcggaaag
     3901 atggccgctt cccggccctg ctgcagggag ctcaaaatgg aggacgcggc gctcgggaga
     3961 gcgggcgggt gagtcaccca cacaaaggaa aagggccttt ccgtcctcag ccgtcgcttc
     4021 atgtgactcc acggagtacc gggcgccgtc caggcacctc gattagttct cgagcttttg
     4081 gagtacgtcg tctttaggtt ggggggaggg gttttatgcg atggagtttc cccacactga
     4141 gtgggtggag actgaagtta ggccagcttg gcacttgatg taattctcct tggaatttgc
     4201 cctttttgag tttggatctt ggttcattct caagcctcag acagtggttc aaagtttttt
     4261 tcttccattt caggtgtcgt gatgtacaat ggccaagttg accagtgccg ttccggtgct
     4321 caccgcgcgc gacgtcgccg gagcggtcga gttctggacc gaccggctcg ggttctcccg
     4381 ggacttcgtg gaggacgact tcgccggtgt ggtccgggac gacgtgaccc tgttcatcag
     4441 cgcggtccag gaccaggtgg tgccggacaa caccctggcc tgggtgtggg tgcgcggcct
     4501 ggacgagctg tacgccgagt ggtcggaggt cgtgtccacg aacttccggg acgcctccgg
     4561 gccggccatg accgagatcg gcgagcagcc gtgggggcgg gagttcgccc tgcgcgaccc
     4621 ggccggcaac tgcgtgcact tcgtggccga ggagcaggac tgagaattcg atatcaagct
     4681 tatcggtaat caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta
     4741 tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc
     4801 ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga
     4861 ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac
     4921 ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc
     4981 cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc
     5041 tcggctgttg ggcactgaca attccgtggt gttgtcgggg aaatcatcgt cctttccttg
     5101 gctgctcgcc tgtgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc
     5161 ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc
     5221 gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcatcg
     5281 ataccgtcga cctcgagacc tagaaaaaca tggagcaatc acaagtagca atacagcagc
     5341 taccaatgct gattgtgcct ggctagaagc acaagaggag gaggaggtgg gttttccagt
     5401 cacacctcag gtacctttaa gaccaatgac ttacaaggca gctgtagatc ttagccactt
     5461 tttaaaagaa aaggggggac tggaagggct aattcactcc caacgaagac aagatatcct
     5521 tgatctgtgg atctaccaca cacaaggcta cttccctgat tggcagaact acacaccagg
     5581 gccagggatc agatatccac tgacctttgg atggtgctac aagctagtac cagttgagca
     5641 agagaaggta gaagaagcca atgaaggaga gaacacccgc ttgttacacc ctgtgagcct
     5701 gcatgggatg gatgacccgg agagagaagt attagagtgg aggtttgaca gccgcctagc
     5761 atttcatcac atggcccgag agctgcatcc ggactgtact gggtctctct ggttagacca
     5821 gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag
     5881 cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag
     5941 atccctcaga cccttttagt cagtgtggaa aatctctagc agggcccgtt taaacccgct
     6001 gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc
     6061 cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg
     6121 catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca
     6181 agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt
     6241 ctgaggcgga aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg
     6301 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     6361 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     6421 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     6481 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     6541 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     6601 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
     6661 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat taattctgtg
     6721 gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     6781 aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg
     6841 cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc
     6901 gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat
     6961 tttttttatt tatgcagagg ccgaggccgc ctctgcctct gagctattcc agaagtagtg
     7021 aggaggcttt tttggaggcc taggcttttg caaaaagctc ccgggagctt gtatatccat
     7081 tttcggatct gatcagcacg tgttgacaat taatcatcgg catagtatat cggcatagta
     7141 taatacgaca aggtgaggaa ctaaaccatg gccaagttga ccagtgccgt tccggtgctc
     7201 accgcgcgcg acgtcgccgg agcggtcgag ttctggaccg accggctcgg gttctcccgg
     7261 gacttcgtgg aggacgactt cgccggtgtg gtccgggacg acgtgaccct gttcatcagc
     7321 gcggtccagg accaggtggt gccggacaac accctggcct gggtgtgggt gcgcggcctg
     7381 gacgagctgt acgccgagtg gtcggaggtc gtgtccacga acttccggga cgcctccggg
     7441 ccggccatga ccgagatcgg cgagcagccg tgggggcggg agttcgccct gcgcgacccg
     7501 gccggcaact gcgtgcactt cgtggccgag gagcaggact gacacgtgct acgagatttc
     7561 gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc
     7621 tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt
     7681 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
     7741 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc
     7801 tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg
     7861 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa
     7921 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
     7981 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     8041 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     8101 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     8161 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     8221 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     8281 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     8341 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     8401 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     8461 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     8521 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     8581 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     8641 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     8701 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     8761 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     8821 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     8881 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     8941 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     9001 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     9061 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     9121 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     9181 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     9241 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     9301 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     9361 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     9421 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     9481 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     9541 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     9601 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     9661 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     9721 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     9781 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     9841 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     9901 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     9961 gttccgcgca catttccccg aaaagtgcca cctgac
//
