LOCUS       sequence-268077-        2275 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  U6 promoter sgRNA entry vector used for all CjeCas9 sgRNAs (clone 
            spacer oligos into BsmBI cassette) - similar to V2-TGAA linker sgRNA
            architecture from Kim et al. Nature Communications 2017.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2275)
  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 2275)
  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-CjeCas9-sgRNA (KAC482)
FEATURES             Location/Qualifiers
     source          1..2275
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        16..264
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     16..36
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     187..206
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        286..356
                     /label=Cj gRNA scaffold
                     /note="guide RNA scaffold for the Campylobacter jejuni 
                     CRISPR/Cas9 system (Kim et al., 2016)"
     rep_origin      complement(460..1048)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(540..559)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1219..2010,2011..2079))
                     /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     1842..1861
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2080..2184)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aaggtcgggc aggaagaggg cctatttccc atgattcctt catatttgca tatacgatac
       61 aaggctgtta gagagataat tagaattaat ttgactgtaa acacaaagat attagtacaa
      121 aatacgtgac gtagaaagta ataatttctt gggtagtttg cagttttaaa attatgtttt
      181 aaaatggact atcatatgct taccgtaact tgaaagtatt tcgatttctt ggctttatat
      241 atcttgtgga aaggacgaaa cacccgagac gattaatgcg tctcggtttt agtccctgaa
      301 gggactaaaa taaagagttt gcgggactct gcggggttac aatcccctaa aaccgctttt
      361 tttaagcttg ggccgctcga ggtacctctc tacatatgac atgtgagcaa aaggccagca
      421 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
      481 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
      541 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
      601 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
      661 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
      721 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
      781 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
      841 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
      901 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
      961 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     1021 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     1081 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     1141 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     1201 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     1261 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     1321 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     1381 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     1441 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     1501 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     1561 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     1621 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     1681 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     1741 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     1801 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     1861 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     1921 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     1981 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     2041 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     2101 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     2161 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtcgctag
     2221 ctgtacaaaa aagcaggctt taaaggaacc aattcagtcg actggatccg gtacc
//
