LOCUS       sequence-193013-        2633 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Empty plasmid backbone to express 2 sgRNAs (use BbsI and BsmBI for 
            cloning).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2633)
  AUTHORS   Billon P, Bryant EE, Joseph SA, Nambiar TS, Hayward SB, Rothstein R,
            Ciccia A
  TITLE     CRISPR-Mediated Base Editing Enables Efficient Disruption of 
            Eukaryotic Genes through Induction of STOP Codons.
  JOURNAL   Mol Cell. 2017 Sep 4. pii: S1097-2765(17)30605-6. doi: 
            10.1016/j.molcel.2017.08.008.
  PUBMED    28890334
REFERENCE   2  (bases 1 to 2633)
  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: "Mol Cell. 
            2017 Sep 4. pii: S1097-2765(17)30605-6. doi: 
            10.1016/j.molcel.2017.08.008."
COMMENT     Sequence Label: B52 (empty plasmid backbone to express 2 sgRNAs)
FEATURES             Location/Qualifiers
     source          1..2633
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        82..330
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        350..425
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        439..687
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        706..781
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     rep_origin      complement(884..1472)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(964..983)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1643..2434,2435..2503))
                     /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     2266..2285
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2504..2608)
                     /gene="bla"
                     /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 gagacgatta atgcgtctcg ttttagagct
      361 agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc
      421 ggtgcttttt ttaagcttga gggcctattt cccatgattc cttcatattt gcatatacga
      481 tacaaggctg ttagagagat aattggaatt aatttgactg taaacacaaa gatattagta
      541 caaaatacgt gacgtagaaa gtaataattt cttgggtagt ttgcagtttt aaaattatgt
      601 tttaaaatgg actatcatat gcttaccgta acttgaaagt atttcgattt cttggcttta
      661 tatatcttgt ggaaaggacg aaacaccggg tcttcgagaa gacctgtttt agagctagaa
      721 atagcaagtt aaaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg
      781 ctttttttct agagaattcc tcgaggtacc tctctacata tgacatgtga gcaaaaggcc
      841 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
      901 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
      961 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     1021 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     1081 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     1141 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     1201 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     1261 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     1321 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     1381 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     1441 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     1501 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     1561 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     1621 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     1681 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     1741 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     1801 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     1861 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     1921 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     1981 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     2041 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     2101 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     2161 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     2221 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     2281 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     2341 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     2401 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     2461 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     2521 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     2581 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cct
//
