LOCUS       sequence-193647-        2636 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  B52 plasmid expressing TIMELESS sgSTOP (cloned in BbsI site) and 
            containing an empty sgRNA-expression cassette (use BsmBI for 
            cloning).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2636)
  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 2636)
  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 + TIMELESS sgSTOP
FEATURES             Location/Qualifiers
     source          1..2636
                     /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        709..784
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     rep_origin      complement(887..1475)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(967..986)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1646..2437,2438..2506))
                     /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     2269..2288
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2507..2611)
                     /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 aaacaccgaa gactcgagcc ctccagcggt tttagagcta
      721 gaaatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg
      781 gtgctttttt tctagagaat tcctcgaggt acctctctac atatgacatg tgagcaaaag
      841 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
      901 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
      961 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     1021 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     1081 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     1141 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     1201 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     1261 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     1321 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     1381 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     1441 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     1501 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     1561 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     1621 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     1681 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     1741 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     1801 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     1861 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     1921 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     1981 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     2041 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     2101 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     2161 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     2221 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     2281 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     2341 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     2401 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     2461 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     2521 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     2581 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacct
//
