LOCUS       40924_41195        2320 bp DNA     circular SYN 13-MAY-2021
DEFINITION  multiplex gRNA expression plasmid.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2320)
  AUTHORS   Tsai SQ, Wyvekens N, Khayter C, Foden JA, Thapar V, Reyon D, Goodwin
            MJ, Aryee MJ, Joung JK
  TITLE     Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome 
            editing.
  JOURNAL   Nat Biotechnol. 2014 Apr 25. doi: 10.1038/nbt.2908.
  PUBMED    24770325
REFERENCE   2  (bases 1 to 2320)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 2320)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Biotechnol. 2014 Apr 25. doi: 10.1038/nbt.2908."
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..2320
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        82..322
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     protein_bind    336..350
                     /label=Csy4 site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease
                     (Haurwitz et al., 2010)"
     misc_RNA        372..447
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    453..467
                     /label=Csy4 site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease
                     (Haurwitz et al., 2010)"
     rep_origin      complement(571..1159)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(1333..2190)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(2191..2295)
                     /label=AmpR promoter
ORIGIN
        1 gacgtcgcta gctgtacaaa aaagcaggct ttaaaggaac caattcagtc gactggatcc
       61 ggtaccaagg tcgggcagga agagggccta tttcccatga ttccttcata tttgcatata
      121 cgatacaagg ctgttagaga gataattaga attaatttga ctgtaaacac aaagatatta
      181 gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta
      241 tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct
      301 ttatatatct tgtggaaagg acgaaacacc gttcactgcc gtataggcag tgagacgatt
      361 aatgcgtctc agttttagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa
      421 cttgaaaaag tggcaccgag tcggtgcgtt cactgccgta taggcagttt ttttaagctt
      481 gggccgctcg aggtacctct ctacatatga catgtgagca aaaggccagc aaaaggccag
      541 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
      601 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
      661 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
      721 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
      781 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
      841 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
      901 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
      961 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     1021 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     1081 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     1141 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     1201 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     1261 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     1321 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     1381 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     1441 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     1501 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     1561 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     1621 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     1681 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     1741 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     1801 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     1861 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     1921 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     1981 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     2041 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     2101 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     2161 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     2221 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     2281 aataggggtt ccgcgcacat ttccccgaaa agtgccacct
//
