LOCUS       sequence-339515-        3401 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Coselection for prime editing in human cells. Vector for tandem 
            expression of ATP1A1 T804N-G6 pegRNA with a user-specified pegRNA 
            from two independent U6 promoters. pU6-pegRNA-GG-acceptor-like 
            plasmid.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3401)
  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 3401)
  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_Dual_pegRNA
FEATURES             Location/Qualifiers
     source          1..3401
                     /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)"
     terminator      complement(259..290)
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
     CDS             complement(307..984)
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     RBS             991..1002
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     protein_bind    1020..1038
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1045..1063
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     rep_origin      complement(1174..1762)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1254..1273)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1933..2724,2725..2793))
                     /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     2556..2575
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2794..2898)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2961..3209
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        3230..3305
                     /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 cgaaacaccc gagaccgtag tcaaaagcct ccggtcggag gcttttgact tggctgagga
      301 gtgccattaa gcaccggtgg agtgacgacc ttcagcacgt tcgtactgtt caacgatggt
      361 gtagtcttcg ttgtgggagg tgatgtccag tttgatgtcg gttttgtaag cacccggcag
      421 ctgaaccggt tttttagcca tgtaggtggt tttaacttca gcgtcgtagt gaccaccgtc
      481 tttcagtttc agacgcattt tgatttcacc tttcagagca ccgtcttccg ggtacatacg
      541 ttcggtggaa gcttcccaac ccatggtttt tttctgcata accggaccgt cggacgggaa
      601 gttggtacca cgcagtttaa ctttgtagat gaactcaccg tcttgcaggg aggagtcctg
      661 ggtaacggta acaacaccac cgtcttcgaa gttcataaca cgttcccatt tgaaaccttc
      721 cgggaaggac agtttcaggt agtccgggat gtcagccggg tgtttaacgt aagctttgga
      781 accgtactgg aactgcgggg acaggatgtc ccaagcgaac ggcagcggac cacctttggt
      841 aactttcagt ttagcggtct gggtaccttc gtacggacga ccttcacctt caccttcgat
      901 ttcgaactcg tgaccgttaa cggaaccttc catacgaact ttgaaacgca tgaactcttt
      961 gataacgtct tcgctactcg ccatagatcc tttctcctct ttcagatccg tgctcagtat
     1021 ctctatcact gatagggatg tcaatctcta tcactgatag ggaggtctcg tttttttaag
     1081 cttgggccgc tcgaggtacc tctctacata tgacatgtga gcaaaaggcc agcaaaaggc
     1141 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     1201 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     1261 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     1321 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     1381 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     1441 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     1501 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     1561 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     1621 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     1681 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     1741 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     1801 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     1861 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     1921 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     1981 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     2041 accatctggc cccagtgctg caatgatacc gcgagatcca cgctcaccgg ctccagattt
     2101 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     2161 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     2221 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     2281 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     2341 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     2401 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     2461 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     2521 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     2581 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     2641 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     2701 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     2761 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     2821 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     2881 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtcg ctagctgtac
     2941 ataccaaggt cgggcaggaa gagggcctat ttcccatgat tccttcatat ttgcatatac
     3001 gatacaaggc tgttagagag ataattagaa ttaatttgac tgtaaacaca aagatattag
     3061 tacaaaatac gtgacgtaga aagtaataat ttcttgggta gtttgcagtt ttaaaattat
     3121 gttttaaaat ggactatcat atgcttaccg taacttgaaa gtatttcgat ttcttggctt
     3181 tatatatctt gtggaaagga cgaaacaccg caaacattcc actaccactg ttttagagct
     3241 agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc
     3301 ggtgcgacat tccccagagg tagtggaatg tttttttaag cttgggccgc tcgaggtacc
     3361 tctctacgtc gactggatcc ggtaccaagg tcgggcagga a
//
