LOCUS       sequence-350192-        2701 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Control vector for coselection for prime editing in human cells. 
            Tandem expression of ATP1A1 T804N-G6 and MTOR-F2108L pegRNAs from 
            two independent U6 promoters..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2701)
  AUTHORS   Levesque S, Mayorga D, Fiset JP, Goupil C, Duringer A, Loiselle A, 
            Bouchard E, Agudelo D, Doyon Y
  TITLE     Marker-free co-selection for successive rounds of prime editing in 
            human cells.
  JOURNAL   Nat Commun. 2022 Oct 7;13(1):5909. doi: 10.1038/s41467-022-33669-z.
  PUBMED    36207338
REFERENCE   2  (bases 1 to 2701)
  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: "Nat 
            Commun."; date: "2022-10-7"; volume: "13(1):5909. doi"; pages: " 
            10.1038/s41467-022-33669-z"
COMMENT     Sequence Label: ATP1A1_G6_T804N_MTOR_F2108L_Dual_pegRNA
FEATURES             Location/Qualifiers
     source          1..2701
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        270..345
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     rep_origin      complement(474..1062)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(554..573)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1233..2024,2025..2093))
                     /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     1856..1875
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2094..2198)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2261..2509
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        2530..2605
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattagaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg cagctgcttt gagattcgtg ttttagagct agaaatagca agttaaaata
      301 aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgctgtgt tgcgacgaat
      361 ctcaaagcag tttttttaag cttgggccgc tcgaggtacc tctctacata tgacatgtga
      421 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
      481 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
      541 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
      601 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
      661 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
      721 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
      781 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
      841 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
      901 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
      961 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     1021 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     1081 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     1141 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     1201 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     1261 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     1321 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagatcca
     1381 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     1441 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     1501 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     1561 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     1621 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     1681 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     1741 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     1801 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     1861 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     1921 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     1981 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     2041 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     2101 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     2161 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     2221 cctgacgtcg ctagctgtac ataccaaggt cgggcaggaa gagggcctat ttcccatgat
     2281 tccttcatat ttgcatatac gatacaaggc tgttagagag ataattagaa ttaatttgac
     2341 tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat ttcttgggta
     2401 gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgcttaccg taacttgaaa
     2461 gtatttcgat ttcttggctt tatatatctt gtggaaagga cgaaacaccg caaacattcc
     2521 actaccactg ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact
     2581 tgaaaaagtg gcaccgagtc ggtgcgacat tccccagagg tagtggaatg tttttttaag
     2641 cttgggccgc tcgaggtacc tctctacgtc gactggatcc ggtaccaagg tcgggcagga
     2701 a
//
