LOCUS       sequence-138996-         950 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  optimized lenti sgRNA cloning backbone with MS2 loops at tetraloop 
            and stemloop 2 and EF1a-puro resistance marker. Contains BsmBI sites
            for insertion of spacer sequences..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 950)
  AUTHORS   Joung J, Konermann S, Gootenberg JS, Abudayyeh OO, Platt RJ, Brigham
            MD, Sanjana NE, Zhang F
  TITLE     Genome-scale CRISPR-Cas9 knockout and transcriptional activation 
            screening.
  JOURNAL   Nat Protoc. 2017 Apr;12(4):828-863. doi: 10.1038/nprot.2017.016. 
            Epub 2017 Mar 23.
  PUBMED    28333914
REFERENCE   2  (bases 1 to 950)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/nprot.2017.016"; journalName: "Nat Protoc"; date: 
            "2017-04"; volume: "12"; issue: "4"; pages: "828-863"
COMMENT     Sequence Label: lenti sgRNA(MS2)_puro optimized backbone
FEATURES             Location/Qualifiers
     source          1..950
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     CDS             complement(54..653)
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(137..157)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     634..653
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     intron          675..878
                     /label=EF-1-alpha intron A
                     /note="truncated intron upstream of the start codon of 
                     human EF-1-alpha"
     primer_bind     complement(698..718)
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
ORIGIN
        1 gtcaatcttt cacaaatttt gtaatccaga ggttgattgt cgacttaacg cgttcaggca
       61 ccgggcttgc gggtcatgca ccaggtgcgc ggtccttcgg gcacctcgac gtcggcggtg
      121 acggtgaagc cgagccgctc gtagaagggg aggttgcggg gcgcggaggt ctccaggaag
      181 gcgggcaccc cggcgcgctc ggccgcctcc actccgggga gcacgacggc gctgcccaga
      241 cccttgccct ggtggtcggg cgagactccg acggtggcca ggaaccacgc gggctccttg
      301 ggccggtgcg gcgccaggag gccttccatc tgttgctgcg cggccagccg ggaaccgctc
      361 aactcggcca tgcgcgggcc gatctcggcg aacaccgccc ccgcttcgac gctctccggc
      421 gtggtccaga ccgccacggc ggcgccgtcg tccgcgaccc acaccttgcc gatgtcgagc
      481 ccgacgcgcg tgaggaagag ttcttgcagc tcggtgaccc gctcgatgtg gcggtccgga
      541 tcgacggtgt ggcgcgtggc ggggtagtcg gcgaacgcgg cggcgagggt gcgtacggcc
      601 ctggggacgt cgtcgcgggt ggcgaggcgc accgtgggct tgtactcggt catggtggcc
      661 gtacgtcacg acacctgaaa tggaagaaaa aaactttgaa ccactgtctg aggcttgaga
      721 atgaaccaag atccaaactc aaaaagggca aattccaagg agaattacat caagtgccaa
      781 gctggcctaa cttcagtctc cacccactca gtgtggggaa actccatcgc ataaaacccc
      841 tccccccaac ctaaagacga cgtactccaa aagctcgaga actaatcgag gtgcctggac
      901 ggcgcccggt actccgtgga gtcacatgaa gcgacggctg aggacggaaa
//
