LOCUS       Exported               10183 bp ds-DNA    circular SYN 06-1-2016
DEFINITION  Zhang lab lentiviral vector, also known as pLentiGuide-Puro, for 
            expressing a single guide RNA (sgRNA) in Cas9-expressing cells. Use 
            together with lentiCas9-Blast.
ACCESSION   .
VERSION     .
KEYWORDS    lentiGuide-Puro
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10183)
  AUTHORS   Sanjana NE, Shalem O, Zhang F.
  TITLE     Improved vectors and genome-wide libraries for CRISPR screening.
  JOURNAL   Nat. Methods 2014;11:783-4.
  PUBMED    25075903
REFERENCE   2  (bases 1 to 10183)
  AUTHORS   Zhang Lab / Addgene #52963
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..10183
                     /organism="synthetic DNA construct"
                     /lab_host="Mammalian Cells"
                     /mol_type="other DNA"
     promoter        87..1265
                     /note="EF-1-alpha promoter"
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          318..1256
                     /note="EF-1-alpha intron A"
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     CDS             1278..1877
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /note="PuroR"
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     misc_feature    1893..2481
                     /note="WPRE"
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     LTR             2553..2786
                     /note="3 LTR (Delta-U3)"
                     /note="self-inactivating 3 long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    2864..2985
                     /note="SV40 poly(A) signal"
                     /note="SV40 polyadenylation signal"
     rep_origin      3025..3160
                     /note="SV40 ori"
                     /note="SV40 origin of replication"
     rep_origin      3367..3822
                     /direction=RIGHT
                     /note="f1 ori"
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        3848..3952
                     /gene="bla"
                     /note="AmpR promoter"
     CDS             3953..4813
                     /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"
     rep_origin      4984..5572
                     /direction=RIGHT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     promoter        6041..6267
                     /note="RSV promoter"
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             6268..6448
                     /note="5 LTR (truncated)"
                     /note="truncated 5 long terminal repeat (LTR) from HIV-1"
     misc_feature    6495..6620
                     /note="HIV-1 Psi"
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    7113..7346
                     /note="RRE"
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     promoter        7753..7993
                     /note="U6 promoter"
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_feature    7998..9882
                     /note="filler"
                     /note="digest with BsmBI to replace the filler with an 
                     sgRNA"
     misc_RNA        9883..9958
                     /note="gRNA scaffold"
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      9959..9964
                     /note="polIII terminator"
                     /note="RNA polymerase III transcription terminator"
     misc_feature    10014..10131
                     /note="cPPT/CTS"
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"