LOCUS Exported 950 bp ds-DNA linear SYN 19-MAY-2021 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 lenti sgRNA(MS2)_puro optimized backbone SOURCE synthetic DNA construct ORGANISM synthetic DNA construct 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 May 19, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..950 /organism="synthetic DNA construct" /mol_type="other DNA" 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 //