LOCUS       sequence-339517-        5652 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 mTOR signaling 
            indicator (TOSI) cassette to ATP1A1 intron 17..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5652)
  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 5652)
  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-TOSI_Donor
FEATURES             Location/Qualifiers
     source          1..5652
                     /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"
     promoter        complement(1049..1559)
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1112..1130)
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     primer_bind     complement(2431..2450)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2550..2572
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2610..2628)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2696..2800
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2801..2869,2870..3661)
                     /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(3019..3038)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3832..4420
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4321..4340
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4574..4591
                     /label=L4440
                     /note="L4440 vector, forward primer"
     polyA_signal    5426..5650
                     /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 tcaccatgga
      781 gccggagtcg gacacgctat cgcctcttcc agaatctcta gagctgttct tccgcagccg
      841 tcttttggcc ttggcgttga ttctggcctc attgatgctg ctggcgctga tccagttgcc
      901 gttgatctcg ttcttaattt cctcggtgcc ggcattttcc tcgtcgccgc tgaacaggct
      961 gtcgctcagg ttgtcaggat cagcggggtt cacgttcagg atctgctcgt tttccacgtc
     1021 catcgtggcc ttaaggctga gggtacccct ggggagagag gtcggtgatt cggtcaacga
     1081 gggagccgac tgccgacgtg cgctccggag gcttgcagaa tgcggaacac cgcgcgggca
     1141 ggaacagggc ccacactacc gccccacacc ccgcctcccg caccgcccct tcccggccgc
     1201 tgctctcggc gcgccctgct gagcagccgc tattggccac agcccatcgc ggtcggcgcg
     1261 ctgccattgc tccctggcgc tgtccgtctg cgagggtact agtgagacgt gcggcttccg
     1321 tttgtcacgt ccggcacgcc gcgaaccgca aggaaccttc ccgacttagg ggcggagcag
     1381 gaagcgtcgc cggggggccc acaagggtag cggcgaagat ccgggtgacg ctgcgaacgg
     1441 acgtgaagaa tgtgcgagac ccagggtcgg cgccgctgcg tttcccggaa ccacgcccag
     1501 agcagccgcg tccctgcgca aacccagggc tgccttggaa aaggcgcaac cccaaccccg
     1561 tggatgcatt aaaaaacctc ccacacctcc ccctgaacct gaaacaagct tgtgaggtgt
     1621 gagctgtgtc ttcattcact ggcactatgc tcccagcatc catgaggctg gcgttgggaa
     1681 aagaaattct caggaccagt atccagtgtg tgtcccaatc ccggcttcac agaatcagta
     1741 ttaccactct tcaaggcagc agttaacatt tgtttatcag atgggaatct ttatttttgt
     1801 atacttcacc taaaagattt ttaaatggga gggcaagaat tttataacaa aaggttcaca
     1861 atattagctt ccttattttt agtaactaaa ttccttctcc ccaccccttc ccaggttcct
     1921 gccatctccc tggcttatga gcaggctgag agtgacatca tgaagagaca gcccagaaat
     1981 cccaaaacag acaaacttgt gaatgagcgg ctgatcagca tggcctatgg gcagattggt
     2041 aagctgcagc ctggagtggg aagctggcac atctaaggca tctgaggtga tggtgtccac
     2101 ctcaggttga ggttgatttc agagactgca aatccaggcg actttcaggt ctaggatgag
     2161 ccctaacgga gtgagcctgt ggagtttctc tgaactctct tccatgtgag aatacatctg
     2221 ttgctgtttg cccagttacc ttttgtggaa actggttttc tatactgagc accttggaac
     2281 tgggctcaac tctgcctgag aaatgaggat gtccttcctc actgagcctt gcggaacagg
     2341 aggcatggtt ctaagggcta gccggaagcg ctcctccctc tgcgagagca atggctccgt
     2401 ccggtttcga gccatatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     2461 ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     2521 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     2581 gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa
     2641 tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg
     2701 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     2761 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     2821 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     2881 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     2941 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     3001 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
     3061 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     3121 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     3181 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     3241 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     3301 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac
     3361 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     3421 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     3481 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
     3541 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     3601 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     3661 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     3721 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     3781 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     3841 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     3901 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     3961 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     4021 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     4081 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     4141 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     4201 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     4261 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     4321 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     4381 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     4441 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     4501 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     4561 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc
     4621 gcctctcccc gcgcgttggc cgattcatta atgcagaatt ccacccacca gagtcattac
     4681 tttgagtagc tcttgttaat gctgggggct atgtttgttg tcacttctca gttctgttat
     4741 ttggtgtagg gcctgtgtga atacttgcct gtgacggttc tcaggcttca taaatagtct
     4801 caataggaaa ggagcagtgt ctgtaatgag tgctcagtgg gggcatgcat cgcactattt
     4861 ccatcgctag gaaaagtgat tggtattaac ccgttttccc ttttctgggg tagggtgcta
     4921 tcgtggctgt gactggtgac ggtgtgaatg actctccagc tttgaagaaa gcagacattg
     4981 gggttgctat ggggattgct ggctcagatg tgtccaagca agctgctgac atgattcttc
     5041 tggatgacaa ctttgcctca attgtgactg gagtagagga aggtgagagc tatttaaggt
     5101 gtacaccaag atcttattca gatactgccc attagcatcc atttctgtat acttcttgga
     5161 tatgttcagt ttccagtgtg cttgtctcat aagctaacag taaaaaatct tggttttcat
     5221 aggtcgtctg atctttgata acttgaagaa atccattgct tataccttaa ccagtaacat
     5281 tcccgagatc accccgttcc tgatatttat tattgcaaac attccactac cactggggaa
     5341 tgtcaccatc ctctgcattg acttgggcac tgacatggtg agtgtcacaa cagtcacaga
     5401 tcgatatcag acgtgatatg tcgacccata gagcccaccg catccccagc atgcctgcta
     5461 ttgtcttccc aatcctcccc cttgctgtcc tgccccaccc caccccccag aatagaatga
     5521 cacctactca gacaatgcga tgcaatttcc tcattttatt aggaaaggac agtgggagtg
     5581 gcaccttcca gggtcaagga aggcacgggg gaggggcaaa caacagatgg ctggcaacta
     5641 gaaggcacag cc
//
