LOCUS       sequence-367467-        3475 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Coselection for prime editing in human cells. Vector for tandem 
            expression of ATP1A1 T804N-G6 epegRNA (tevopreQ1) with a 
            user-specified epegRNA. pU6-tevopreq1-GG-acceptor-like plasmid.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3475)
  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 3475)
  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_v1_Dual_epegRNA_tevopreQ1
FEATURES             Location/Qualifiers
     source          1..3475
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        38..286
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        307..382
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        516..764
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     terminator      complement(774..805)
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
     CDS             complement(822..1499)
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     RBS             1506..1517
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     protein_bind    1535..1553
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1560..1578
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     rep_origin      complement(1726..2314)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1806..1825)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2485..3276,3277..3345))
                     /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     3108..3127
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3346..3450)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacgtcgcta gctgtacata ccaaggtcgg gcaggaagag ggcctatttc ccatgattcc
       61 ttcatatttg catatacgat acaaggctgt tagagagata attagaatta atttgactgt
      121 aaacacaaag atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt
      181 tgcagtttta aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta
      241 tttcgatttc ttggctttat atatcttgtg gaaaggacga aacaccgcaa acattccact
      301 accactgttt tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga
      361 aaaagtggca ccgagtcggt gcgacattcc ccagaggtag tggaatgcgc ggttctatct
      421 agttacgcgt taaaccaact agaatttttt taagcttggg ccgctcgagg tacctctcta
      481 cgtcgactgg atccggtacc aaggtcgggc aggaagaggg cctatttccc atgattcctt
      541 catatttgca tatacgatac aaggctgtta gagagataat tagaattaat ttgactgtaa
      601 acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg
      661 cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt
      721 tcgatttctt ggctttatat atcttgtgga aaggacgaaa cacccgagac cgtagtcaaa
      781 agcctccggt cggaggcttt tgacttggct gaggagtgcc attaagcacc ggtggagtga
      841 cgaccttcag cacgttcgta ctgttcaacg atggtgtagt cttcgttgtg ggaggtgatg
      901 tccagtttga tgtcggtttt gtaagcaccc ggcagctgaa ccggtttttt agccatgtag
      961 gtggttttaa cttcagcgtc gtagtgacca ccgtctttca gtttcagacg cattttgatt
     1021 tcacctttca gagcaccgtc ttccgggtac atacgttcgg tggaagcttc ccaacccatg
     1081 gtttttttct gcataaccgg accgtcggac gggaagttgg taccacgcag tttaactttg
     1141 tagatgaact caccgtcttg cagggaggag tcctgggtaa cggtaacaac accaccgtct
     1201 tcgaagttca taacacgttc ccatttgaaa ccttccggga aggacagttt caggtagtcc
     1261 gggatgtcag ccgggtgttt aacgtaagct ttggaaccgt actggaactg cggggacagg
     1321 atgtcccaag cgaacggcag cggaccacct ttggtaactt tcagtttagc ggtctgggta
     1381 ccttcgtacg gacgaccttc accttcacct tcgatttcga actcgtgacc gttaacggaa
     1441 ccttccatac gaactttgaa acgcatgaac tctttgataa cgtcttcgct actcgccata
     1501 gatcctttct cctctttcag atccgtgctc agtatctcta tcactgatag ggatgtcaat
     1561 ctctatcact gatagggagg tctcgcgcgg ttctatctag ttacgcgtta aaccaactag
     1621 aattttttta agcttgggcc gctcgaggta cctctctaca tatgacatgt gagcaaaagg
     1681 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     1741 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     1801 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     1861 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     1921 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     1981 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     2041 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     2101 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     2161 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     2221 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     2281 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     2341 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     2401 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     2461 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     2521 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     2581 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagatc cacgctcacc
     2641 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     2701 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     2761 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     2821 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     2881 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     2941 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     3001 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     3061 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     3121 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     3181 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     3241 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     3301 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     3361 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     3421 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacct
//
