LOCUS       sequence-171421-        3217 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Homology-directed repair (HDR) donor plasmid with homology arms to 
            ATP1A1 to introduce the Q118R and N129D (RD) mutations conferring 
            cellular resistance to ouabain.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3217)
  AUTHORS   Agudelo D, Duringer A, Bozoyan L, Huard CC, Carter S, Loehr J, 
            Synodinou D, Drouin M, Salsman J, Dellaire G, Laganiere J, Doyon Y
  TITLE     Marker-free coselection for CRISPR-driven genome editing in human 
            cells.
  JOURNAL   Nat Methods. 2017 Apr 17. doi: 10.1038/nmeth.4265.
  PUBMED    28417998
REFERENCE   2  (bases 1 to 3217)
  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 
            Methods. 2017 Apr 17. doi: 10.1038/nmeth.4265."
COMMENT     Sequence Label: ATP1A1_plasmid_donor_RD
FEATURES             Location/Qualifiers
     source          1..3217
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1227..1244)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1398..1986)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1478..1497)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2157..2948,2949..3017))
                     /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     2780..2799
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3018..3122)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3190..3208
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatggt accaaattta ttgatggatc aatttaaaga gttttaatct gggtgttatg
      241 agttccttgg gcctattgtt tgcctgaacc ctgtggggac tggctcatca gcagaattat
      301 tcatggagga atttgctagg ttttaccttg gctctctagc ttgggacatt ttgtttcttc
      361 cttaaatcct tattgcaacc gtccagctac caggtaggta tattgccttg taagtgctgg
      421 tacagtttgc cttatttata ttccactgct tctcagggat taacatctgc tcgtgcagct
      481 gagatcctgg cgcgagatgg tcccaacgcc ctcactcccc ctcccactac tcctgaatgg
      541 atcaagtttt gtcggcagct ctttgggggg ttctcaatgt tactgtggat tggagcgatt
      601 ctttgtttct tggcttatag catcagagct gctacagaag aggaacctca aaacgatgac
      661 gtgagttctg taattcagca tatcgatttg tagtacacat cagatatctt ctccgtcttt
      721 gtctcccact tcttctcaat taccactcat tacttaatgg ttatgaactc attacttaat
      781 ggttatgaac agctgttgcc ttcaaggctc atccattctt ccttcgtttc catttcctct
      841 ctctaccacc cacgttgtag atgctcttac aagtgggatg cccacctgca tgtgctgctg
      901 tgagcaggca gctctgctca ggccccggcc gccacccact ggatggcaga gcacagcgat
      961 tcatgttggc acatccacct gtccagaaat gcttggtggc cattcttcaa aatccacaag
     1021 ttggttgaaa aacaatcttt gttttataaa gcaggagaaa ctgatgcatc tagaaccttt
     1081 ccaaacgtcc agttagtgat caagtgttgg tgtgcctgac tccatttctg acccttcctg
     1141 tgtggtcttt agaagggaat tctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     1201 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     1261 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     1321 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     1381 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     1441 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     1501 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     1561 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     1621 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     1681 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     1741 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     1801 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     1861 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     1921 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     1981 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     2041 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     2101 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     2161 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     2221 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     2281 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     2341 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     2401 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     2461 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     2521 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     2581 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     2641 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     2701 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     2761 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     2821 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     2881 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     2941 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     3001 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     3061 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     3121 cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta
     3181 acctataaaa ataggcgtat cacgaggccc tttcgtc
//
