LOCUS       sequence-339523-        5433 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  HDR donor plasmid to introduce the T804N mutation conferring 
            cellular resistance to ouabain and the mScarlet-I cassette to ATP1A1
            intron 17..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5433)
  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 5433)
  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_T804N_hPGK1_mScarlet-I_Donor
FEATURES             Location/Qualifiers
     source          1..5433
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(34..81)
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             complement(82..777)
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet-I
                     /note="matures faster than mScarlet"
                     /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFSWDILSPQFMYGSRAFIKHPADIPDYYKQSFPEGFKWERVMNFEDGG
                     AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK
                     MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH
                     STGGMDELYK"
     primer_bind     complement(357..376)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     626..644
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             complement(781..801)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     promoter        complement(830..1340)
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     complement(893..911)
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     primer_bind     complement(2212..2231)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2331..2353
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2391..2409)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2477..2581
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2582..2650,2651..3442)
                     /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     complement(2800..2819)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3613..4201
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4102..4121
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4355..4372
                     /label=L4440
                     /note="L4440 vector, forward primer"
     polyA_signal    5207..5431
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
ORIGIN
        1 tgcagggttt aaacgcggcc gctcgagtta tcactttttc ttttttgcct ggccggcctt
       61 tttcgtggcc gccggccttt tcttgtacag ctcgtccatg ccgccggtgg agtggcggcc
      121 ctcggagcgt tcgtactgtt ccaccacggt gtagtcctcg ttgtgggagg tgatgtccaa
      181 cttgcggtcg acgttgtagg cgccgggcat ctgcacgggc ttcttggcct tgtaggtggt
      241 cttgaagtcc gccaggtagc ggccgccgtc cttcaggcgc agggccatct taatgtcgcc
      301 cttcagcacg ccgtcctcgg ggtacaaccg ctcggtggat gcttcccagc ccattgtctt
      361 cttctgcatt acggggccgt caggagggaa gttggtgccg cggagcttca ccttgtagat
      421 cagggtgccg tcctccaggg aggtgtcctg ggtcacggtc acggcgccgc cgtcctcgaa
      481 gttcatcacg cgctcccact tgaagccctc ggggaaggac tgcttatagt agtcggggat
      541 gtcggcgggg tgcttgatga aggccctgga gccgtacatg aactgagggg acaggatgtc
      601 ccaggagaag ggcagggggc cacccttggt caccttcagc ttggcggtct gggtgccctc
      661 gtaggggcgg ccctcgccct cgccctcgat ctcgaactcg tggccgttca tggagccctc
      721 catgtgcacc ttgaaccgca tgaactcctt gatcactgcc tcgcccttgc tcaccatggc
      781 tccctgaaaa tacagattct cggcggccgc cttaaggctg agggtacccc tggggagaga
      841 ggtcggtgat tcggtcaacg agggagccga ctgccgacgt gcgctccgga ggcttgcaga
      901 atgcggaaca ccgcgcgggc aggaacaggg cccacactac cgccccacac cccgcctccc
      961 gcaccgcccc ttcccggccg ctgctctcgg cgcgccctgc tgagcagccg ctattggcca
     1021 cagcccatcg cggtcggcgc gctgccattg ctccctggcg ctgtccgtct gcgagggtac
     1081 tagtgagacg tgcggcttcc gtttgtcacg tccggcacgc cgcgaaccgc aaggaacctt
     1141 cccgacttag gggcggagca ggaagcgtcg ccggggggcc cacaagggta gcggcgaaga
     1201 tccgggtgac gctgcgaacg gacgtgaaga atgtgcgaga cccagggtcg gcgccgctgc
     1261 gtttcccgga accacgccca gagcagccgc gtccctgcgc aaacccaggg ctgccttgga
     1321 aaaggcgcaa ccccaacccc gtggatgcat taaaaaacct cccacacctc cccctgaacc
     1381 tgaaacaagc ttgtgaggtg tgagctgtgt cttcattcac tggcactatg ctcccagcat
     1441 ccatgaggct ggcgttggga aaagaaattc tcaggaccag tatccagtgt gtgtcccaat
     1501 cccggcttca cagaatcagt attaccactc ttcaaggcag cagttaacat ttgtttatca
     1561 gatgggaatc tttatttttg tatacttcac ctaaaagatt tttaaatggg agggcaagaa
     1621 ttttataaca aaaggttcac aatattagct tccttatttt tagtaactaa attccttctc
     1681 cccacccctt cccaggttcc tgccatctcc ctggcttatg agcaggctga gagtgacatc
     1741 atgaagagac agcccagaaa tcccaaaaca gacaaacttg tgaatgagcg gctgatcagc
     1801 atggcctatg ggcagattgg taagctgcag cctggagtgg gaagctggca catctaaggc
     1861 atctgaggtg atggtgtcca cctcaggttg aggttgattt cagagactgc aaatccaggc
     1921 gactttcagg tctaggatga gccctaacgg agtgagcctg tggagtttct ctgaactctc
     1981 ttccatgtga gaatacatct gttgctgttt gcccagttac cttttgtgga aactggtttt
     2041 ctatactgag caccttggaa ctgggctcaa ctctgcctga gaaatgagga tgtccttcct
     2101 cactgagcct tgcggaacag gaggcatggt tctaagggct agccggaagc gctcctccct
     2161 ctgcgagagc aatggctccg tccggtttcg agccatatgg tgcactctca gtacaatctg
     2221 ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg
     2281 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
     2341 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat
     2401 acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac
     2461 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     2521 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     2581 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     2641 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     2701 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     2761 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     2821 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     2881 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     2941 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     3001 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     3061 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     3121 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     3181 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     3241 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     3301 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     3361 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     3421 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     3481 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     3541 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     3601 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     3661 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     3721 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     3781 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     3841 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     3901 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     3961 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     4021 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     4081 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     4141 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     4201 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     4261 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     4321 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     4381 agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagaat
     4441 tccacccacc agagtcatta ctttgagtag ctcttgttaa tgctgggggc tatgtttgtt
     4501 gtcacttctc agttctgtta tttggtgtag ggcctgtgtg aatacttgcc tgtgacggtt
     4561 ctcaggcttc ataaatagtc tcaataggaa aggagcagtg tctgtaatga gtgctcagtg
     4621 ggggcatgca tcgcactatt tccatcgcta ggaaaagtga ttggtattaa cccgttttcc
     4681 cttttctggg gtagggtgct atcgtggctg tgactggtga cggtgtgaat gactctccag
     4741 ctttgaagaa agcagacatt ggggttgcta tggggattgc tggctcagat gtgtccaagc
     4801 aagctgctga catgattctt ctggatgaca actttgcctc aattgtgact ggagtagagg
     4861 aaggtgagag ctatttaagg tgtacaccaa gatcttattc agatactgcc cattagcatc
     4921 catttctgta tacttcttgg atatgttcag tttccagtgt gcttgtctca taagctaaca
     4981 gtaaaaaatc ttggttttca taggtcgtct gatctttgat aacttgaaga aatccattgc
     5041 ttatacctta accagtaaca ttcccgagat caccccgttc ctgatattta ttattgcaaa
     5101 cattccacta ccactgggga atgtcaccat cctctgcatt gacttgggca ctgacatggt
     5161 gagtgtcaca acagtcacag atcgatatca gacgtgatat gtcgacccat agagcccacc
     5221 gcatccccag catgcctgct attgtcttcc caatcctccc ccttgctgtc ctgccccacc
     5281 ccacccccca gaatagaatg acacctactc agacaatgcg atgcaatttc ctcattttat
     5341 taggaaagga cagtgggagt ggcaccttcc agggtcaagg aaggcacggg ggaggggcaa
     5401 acaacagatg gctggcaact agaaggcaca gcc
//
