LOCUS       sequence-82647-e        2735 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  sgRNA expression construction in a 3rd generation lentiviral 
            backbone.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 2735)
  AUTHORS   Zhou Y, Zhu S, Cai C, Yuan P, Li C, Huang Y, Wei W
  TITLE     High-throughput screening of a CRISPR/Cas9 library for functional 
            genomics in human cells.
  JOURNAL   Nature. 2014 Apr 9. doi: 10.1038/nature13166.
  PUBMED    24717434
REFERENCE   2  (bases 1 to 2735)
  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"; journalName: "Nature. 
            2014 Apr 9. doi: 10.1038/nature13166."
COMMENT     Sequence Label: pLenti-sgRNA-Lib
FEATURES             Location/Qualifiers
     source          1..2735
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     promoter        28..276
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     28..48
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     199..218
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     300..319
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        300..318
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    348..472
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(901..1206)
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     complement(1003..1022)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(1526..2209)
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGRKRR
                     VDPAY"
     primer_bind     2124..2143
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(2210..2312)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     protein_bind    complement(2490..2614)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     misc_RNA        2647..2722
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
ORIGIN
        1 tctagaggta ccaaggtctg gcaggaagag ggcctatttc ccatgattcc ttcatatttg
       61 catatacgat acaaggctgt tagagagata attagaatta atttgactgt aaacacaaag
      121 atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt tgcagtttta
      181 aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta tttcgatttc
      241 ttggctttat atatcttgtg gaaaggacga aacaccgaga gacgctggct tatcgaaatt
      301 aatacgactc actataggga gacccaagct ggctagttaa gctatcaaca agtttgtaca
      361 aaaaagctga acgagaaacg taaaatgata taaatatcaa tatattaaat tagattttgc
      421 ataaaaaaca gactacataa tactgtaaaa cacaacatat ccagtcacta tgaatcaact
      481 acttagatgg tattagtgac ctgtagtcga ccgacagcct tccaaatgtt cttcgggtga
      541 tgctgccaac ttagtcgacc gacagccttc caaatgttct tctcaaacgg aatcgtcgta
      601 tccagcctac tcgctattgt cctcaatgcc gtattaaatc ataaaaagaa ataagaaaaa
      661 gaggtgcgag cctctttttt gtgtgacaaa ataaaaacat ctacctattc atatacgcta
      721 gtgtcatagt cctgaaaatc atctgcatca agaacaattt cacaactctt atacttttct
      781 cttacaagtc gttcggcttc atctggattt tcagcctcta tacttactaa acgtgataaa
      841 gtttctgtaa tttctactgt atcgacctgc agactggctg tgtataaggg agcctgacat
      901 ttatattccc cagaacatca ggttaatggc gtttttgatg tcattttcgc ggtggctgag
      961 atcagccact tcttccccga taacggagac cggcacactg gccatatcgg tggtcatcat
     1021 gcgccagctt tcatccccga tatgcaccac cgggtaaagt tcacgggaga ctttatctga
     1081 cagcagacgt gcactggcca gggggatcac catccgtcgc ccgggcgtgt caataatatc
     1141 actctgtaca tccacaaaca gacgataacg gctctctctt ttataggtgt aaaccttaaa
     1201 ctgcatttca ccagcccctg ttctcgtcag caaaagagcc gttcatttca ataaaccggg
     1261 cgacctcagc catcccttcc tgattttccg ctttccagcg ttcggcacgc agacgacggg
     1321 cttcattctg catggttgtg cttaccagac cggagatatt gacatcatat atgccttgag
     1381 caactgatag ctgtcgctgt caactgtcac tgtaatacgc tgcttcatag catacctctt
     1441 tttgacatac ttcgggtata catatcagta tatattctta taccgcaaaa atcagcgcgc
     1501 aaatacgcat actgttatct ggcttttagt aagccggatc cacgcggcgt ttacgccccc
     1561 cctgccactc atcgcagtac tgttgtaatt cattaagcat tctgccgaca tggaagccat
     1621 cacaaacggc atgatgaacc tgaatcgcca gcggcatcag caccttgtcg ccttgcgtat
     1681 aatatttgcc catggtgaaa acgggggcga agaagttgtc catattggcc acgtttaaat
     1741 caaaactggt gaaactcacc cagggattgg ctgagacgaa aaacatattc tcaataaacc
     1801 ctttagggaa ataggccagg ttttcaccgt aacacgccac atcttgcgaa tatatgtgta
     1861 gaaactgccg gaaatcgtcg tggtattcac tccagagcga tgaaaacgtt tcagtttgct
     1921 catggaaaac ggtgtaacaa gggtgaacac tatcccatat caccagctca ccgtctttca
     1981 ttgccatacg gaattccgga tgagcattca tcaggcgggc aagaatgtga ataaaggccg
     2041 gataaaactt gtgcttattt ttctttacgg tctttaaaaa ggccgtaata tccagctgaa
     2101 cggtctggtt ataggtacat tgagcaactg actgaaatgc ctcaaaatgt tctttacgat
     2161 gccattggga tatatcaacg gtggtatatc cagtgatttt tttctccatt ttagcttcct
     2221 tagctcctga aaatctcgat aactcaaaaa atacgcccgg tagtgatctt atttcattat
     2281 ggtgaaagtt ggaacctctt acgtgccgat caacgtctca ttttcgccaa aagttggccc
     2341 agggcttccc ggtatcaaca gggacaccag gatttattta ttctgcgaag tgatcttccg
     2401 tcacaggtat ttattcggcg caaagtgcgt cgggtgatgc tgccaactta gtcgactaca
     2461 ggtcactaat accatctaag tagttgattc atagtgactg gatatgttgt gttttacagt
     2521 attatgtagt ctgtttttta tgcaaaatct aatttaatat attgatattt atatcatttt
     2581 acgtttctcg ttcagctttc ttgtacaaag tggttgatct agagggcccg cggttcgaac
     2641 gtctctgttt tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga
     2701 aaaagtggca ccgagtcggt gctttttttc tcgag
//
