LOCUS       sequence-259726-        2305 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  S. pyogenes pegRNA for CTT insertion at the HEK3 site of human cells
            using prime editing.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2305)
  AUTHORS   Anzalone AV, Randolph PB, Davis JR, Sousa AA, Koblan LW, Levy JM, 
            Chen PJ, Wilson C, Newby GA, Raguram A, Liu DR
  TITLE     Search-and-replace genome editing without double-strand breaks or 
            donor DNA.
  JOURNAL   Nature. 2019 Oct 21. pii: 10.1038/s41586-019-1711-4. doi: 
            10.1038/s41586-019-1711-4.
  PUBMED    31634902
REFERENCE   2  (bases 1 to 2305)
  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. 
            2019 Oct 21. pii: 10.1038/s41586-019-1711-4. doi: 
            10.1038/s41586-019-1711-4."
COMMENT     Sequence Label: pU6-Sp-pegRNA-HEK3_CTT_ins
FEATURES             Location/Qualifiers
     source          1..2305
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        29..104
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     rep_origin      complement(234..822)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(314..333)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(993..1784,1785..1853))
                     /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     1616..1635
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1854..1958)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        join(2065..2305,1..8)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2065..2085
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2236..2255
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
ORIGIN
        1 gaaacaccgg cccagactga gcacgtgagt tttagagcta gaaatagcaa gttaaaataa
       61 ggctagtccg ttatcaactt gaaaaagtgg gaccgagtcg gtcctctgcc atcaaagcgt
      121 gctcagtctg tttttttaag cttgggccgc tcgaggtacc tctctacata tgacatgtga
      181 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
      241 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
      301 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
      361 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
      421 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
      481 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
      541 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
      601 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
      661 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
      721 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
      781 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
      841 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
      901 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
      961 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     1021 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     1081 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     1141 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     1201 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     1261 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     1321 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     1381 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     1441 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     1501 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     1561 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     1621 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     1681 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     1741 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     1801 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     1861 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     1921 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     1981 cctgacgtcg ctagctgtac aaaaaagcag gctttaaagg aaccaattca gtcgactgga
     2041 tccggtacca aggtcgggca ggaagagggc ctatttccca tgattccttc atatttgcat
     2101 atacgataca aggctgttag agagataatt agaattaatt tgactgtaaa cacaaagata
     2161 ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa
     2221 ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg
     2281 gctttatata tcttgtggaa aggac
//
