LOCUS       sequence-268589-        2296 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  U6 promoter sgRNA entry vector used for all St3Cas9 sgRNAs (clone 
            spacer oligos into BsmBI cassette) - similar to sgRNA architecture 
            from Muller et al. Molecular Therapy 2015.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2296)
  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 2296)
  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-St3Cas9-sgRNA (KAC27)
FEATURES             Location/Qualifiers
     source          1..2296
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        complement(806..1054)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(864..883)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(1034..1054)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     promoter        1161..1265
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1266..1334,1335..2126)
                     /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     complement(1484..1503)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt catatgtaga
      661 gaggtacctc gagcggccca agcttaaaaa aacaccgaat cggtgccacc ttttcaagtt
      721 gagtacggac taagccttat tttaactcgc tgtgttgttt taacaaacaa cacagctcta
      781 aaaccgagac gcattaatcg tctcgggtgt ttcgtccttt ccacaagata tataaagcca
      841 agaaatcgaa atactttcaa gttacggtaa gcatatgata gtccatttta aaacataatt
      901 ttaaaactgc aaactaccca agaaattatt actttctacg tcacgtattt tgtactaata
      961 tctttgtgtt tacagtcaaa ttaattctaa ttatctctct aacagccttg tatcgtatat
     1021 gcaaatatga aggaatcatg ggaaataggc cctcttcctg cccgaccttg gtaccggatc
     1081 cagtcgactg aattggttcc tttaaagcct gcttttttgt acagctagcg acgtcaggtg
     1141 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     1201 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     1261 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     1321 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     1381 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     1441 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     1501 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     1561 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     1621 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     1681 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     1741 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     1801 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     1861 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     1921 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     1981 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     2041 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     2101 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     2161 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     2221 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     2281 agatcaaagg atcttc
//
