LOCUS       sequence-339508-        3003 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Coselection for ABE or NHEJ in human cells. Vector for tandem 
            expression of ATP1A1 G2 sgRNA with a user-specified sgRNA from two 
            independent U6 promoters. Cloning of oligos using BbsI sites..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3003)
  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 3003)
  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_G2_Dual_sgRNA
FEATURES             Location/Qualifiers
     source          1..3003
                     /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        268..343
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        462..710
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        731..806
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(832..848)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(832..848)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(845..867)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    856..872
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(880..886,887..904,905..910))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    925..946
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1063..1080)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1234..1822)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1314..1333)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1993..2784,2785..2853))
                     /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     2616..2635
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2854..2958)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg ggtcttcgag aagacctgtt ttagagctag aaatagcaag ttaaaataag
      301 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg ttttagagct
      361 agaaatagca agttaaaata aggctagtcc gtttttagcg cgtgcgccaa ttctgcagac
      421 aaatggcgtc gactggatcc ggtaccaagg tcgggcagga agagggccta tttcccatga
      481 ttccttcata tttgcatata cgatacaagg ctgttagaga gataattaga attaatttga
      541 ctgtaaacac aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt
      601 agtttgcagt tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa
      661 agtatttcga tttcttggct ttatatatct tgtggaaagg acgaaacacc gatccaagct
      721 gctacagaag gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac
      781 ttgaaaaagt ggcaccgagt cggtgctttt tttaagcttg gcgtaatcat ggtcatagct
      841 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
      901 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
      961 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     1021 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     1081 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     1141 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     1201 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     1261 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     1321 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     1381 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     1441 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     1501 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     1561 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     1621 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     1681 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     1741 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     1801 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     1861 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     1921 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     1981 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     2041 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     2101 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     2161 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     2221 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     2281 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     2341 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     2401 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     2461 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     2521 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     2581 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     2641 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     2701 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     2761 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     2821 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     2881 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     2941 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtcg ctagctgtac
     3001 agt
//
