LOCUS       Exported                9996 bp ds-DNA    circular SYN 06-1-2016
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
FEATURES             Location/Qualifiers
     source          1..9996
                     /organism="synthetic DNA construct"
                     /lab_host="Mammalian Cells"
                     /mol_type="other DNA"
     enhancer        238..617
                     /note="CMV enhancer"
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        619..817
                     /note="CMV promoter"
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     LTR             835..1015
                     /note="5 LTR (truncated)"
                     /note="truncated 5 long terminal repeat (LTR) from HIV-1"
     misc_feature    1062..1187
                     /note="HIV-1 Psi"
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1680..1913
                     /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    2440..2557
                     /note="cPPT/CTS"
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2607..2847
                     /note="U6 promoter"
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_RNA        2876..3011
                     /note="gRNA scaffold"
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system, modified by the insertion of two MS2 
                     stem loops"
     misc_RNA        2893..2911
                     /note="MS2 stem loop"
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        2963..2981
                     /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
                     /note="EF-1-alpha promoter"
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          3336..4273
                     /note="EF-1-alpha intron A"
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     CDS             4289..4663
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /note="BleoR"
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     misc_feature    4688..5276
                     /note="WPRE"
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     LTR             5801..5981
                     /note="5 LTR (truncated)"
                     /note="truncated 5 long terminal repeat (LTR) from HIV-1"
     polyA_signal    6013..6237
                     /note="bGH poly(A) signal"
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      6283..6711
                     /direction=RIGHT
                     /note="f1 ori"
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        6725..7054
                     /note="SV40 promoter"
                     /note="SV40 enhancer and early promoter"
     rep_origin      6905..7040
                     /note="SV40 ori"
                     /note="SV40 origin of replication"
     promoter        7102..7149
                     /note="EM7 promoter"
                     /note="synthetic bacterial promoter "
     CDS             7168..7542
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /note="BleoR"
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    7672..7793
                     /note="SV40 poly(A) signal"
                     /note="SV40 polyadenylation signal"
     rep_origin      complement(8244..8832)
                     /direction=LEFT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(9003..9863)
                     /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(9864..9968)
                     /gene="bla"
                     /note="AmpR promoter"