LOCUS       sequence-451024-        2255 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pU6 entry plamid for cloning prime editor mpknot epegRNAs (requires 
            BsmBI or Esp3I digest and ligation of duplexed oligos).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2255)
  AUTHORS   Silverstein RA, Kim N, Kroell AS, Walton RT, Delano J, Butcher RM, 
            Pacesa M, Smith BK, Christie KA, Ha LL, Meis RJ, Clark AB, Spinner 
            AD, Lazzarotto CR, Li Y, Matsubara A, Urbina EO, Dahl GA, Correia 
            BE, Marks DS, Tsai SQ, Pinello L, De Ravin SS, Liu Q, Kleinstiver BP
  TITLE     Custom CRISPR-Cas9 PAM variants via scalable engineering and machine
            learning.
  JOURNAL   Nature. 2025 Apr 22. doi: 10.1038/s41586-025-09021-y.
  PUBMED    40262634
REFERENCE   2  (bases 1 to 2255)
  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: "Nature. 
            2025 Apr 22. doi: 10.1038/s41586-025-09021-y."
COMMENT     Sequence Label: pUC19-U6-BsmBI_epegRNA_entry-mpknot_(LM1140)
FEATURES             Location/Qualifiers
     source          1..2255
                     /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)"
     primer_bind     82..102
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     253..272
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     rep_origin      complement(506..1094)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(586..605)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1265..2056,2057..2125))
                     /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     1888..1907
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2126..2230)
                     /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 ggagacgatt aatgcgtctc cgggtcagga
      361 gccccccccc tgaacccagg ataaccctca aagtcggggg gcttttttta agcttgggcc
      421 gctcgaggta cctctctaca tatgacatgt gagcaaaagg ccagcaaaag gccaggaacc
      481 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
      541 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
      601 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
      661 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
      721 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
      781 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
      841 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
      901 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
      961 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     1021 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     1081 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     1141 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     1201 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     1261 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     1321 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     1381 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     1441 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     1501 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     1561 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     1621 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     1681 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     1741 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     1801 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     1861 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     1921 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     1981 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     2041 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     2101 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     2161 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     2221 gggttccgcg cacatttccc cgaaaagtgc cacct
//
