LOCUS       sequence-268076-        2321 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  U6 promoter sgRNA entry vector used for all NmeCas9 or Nme2Cas9 
            sgRNAs (clone spacer oligos into BsmBI cassette) - similar to sgRNA 
            architecture from Amrani et al. Genome Biology 2018.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2321)
  AUTHORS   Mahendra C, Christie KA, Osuna BA, Pinilla-Redondo R, Kleinstiver 
            BP, Bondy-Denomy J
  TITLE     Broad-spectrum anti-CRISPR proteins facilitate horizontal gene 
            transfer.
  JOURNAL   Nat Microbiol. 2020 Apr;5(4):620-629. doi: 
            10.1038/s41564-020-0692-2. Epub 2020 Mar 26.
  PUBMED    32218510
REFERENCE   2  (bases 1 to 2321)
  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/s41564-020-0692-2"; journalName: "Nat Microbiol"; date: 
            "2020-04"; volume: "5"; issue: "4"; pages: "620-629"
COMMENT     Sequence Label: pUC19-U6-BsmBI_cassette-Nme/Nme2Cas9-sgRNA (KA...
FEATURES             Location/Qualifiers
     source          1..2321
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1..21
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     172..191
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        299..390
                     /label=Nm tracrRNA
                     /note="trans-activating CRISPR RNA for the Neisseria 
                     meningitidis CRISPR/Cas9 system (Hou et al., 2013)"
     rep_origin      complement(491..1079)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(571..590)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1250..2041,2042..2110))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     1873..1892
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2111..2215)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattagaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccc gagacgatta atgcgtctcg gttgtagctc cctttctcat ttcggaaacg
      301 aaatgagaac cgttgctaca ataaggccgt ctgaaaagat gtgccgcaac gctctgcccc
      361 ttaaagcttc tgctttaagg ggcatcgttt ttttaagctt gggccgctcg aggtacctct
      421 ctacatatga catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
      481 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
      541 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
      601 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
      661 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
      721 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
      781 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
      841 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
      901 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
      961 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     1021 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     1081 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     1141 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     1201 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     1261 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     1321 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     1381 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     1441 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     1501 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     1561 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     1621 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     1681 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     1741 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     1801 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     1861 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     1921 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     1981 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     2041 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     2101 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     2161 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     2221 ttccccgaaa agtgccacct gacgtcgcta gctgtacaaa aaagcaggct ttaaaggaac
     2281 caattcagtc gactggatcc ggtaccaagg tcgggcagga a
//
