LOCUS       sequence-367518-        5382 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  HDR donor plasmid to introduce the T804N mutation conferring 
            cellular resistance to ouabain and the EBFP cassette to ATP1A1 
            intron 17..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5382)
  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 5382)
  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_EBFP_Donor
FEATURES             Location/Qualifiers
     source          1..5382
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(7..720,721..723,724..726))
                     /codon_start=1
                     /product="enhanced blue variant of GFP"
                     /label=EBFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSHGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(52..73)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     401..420
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     660..681
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(730..750)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     promoter        complement(779..1289)
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     complement(842..860)
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     primer_bind     complement(2161..2180)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2280..2302
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2340..2358)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2426..2530
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2531..2599,2600..3391)
                     /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(2749..2768)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3562..4150
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4051..4070
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4304..4321
                     /label=L4440
                     /note="L4440 vector, forward primer"
     polyA_signal    5156..5380
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
ORIGIN
        1 tgcaggtcac ttgtacagct cgtccatgcc gagagtgatc ccggcggcgg tcacgaactc
       61 cagcaggacc atgtgatcgc gcttctcgtt ggggtctttg ctcagggcgg actgggtgct
      121 caggtagtgg ttgtcgggca gcagcacggg gccgtcgccg atgggggtgt tctgctggta
      181 gtggtcggcg agctgcacgc tgccgtcctc gatgttgtgg cggatcttga agttcacctt
      241 gatgccgttc ttctgcttgt cggccatgat atagacgttg tggctgttgt agttgtactc
      301 cagcttgtgc cccaggatgt tgccgtcctc cttgaagtcg atgcccttca gctcgatgcg
      361 gttcaccagg gtgtcgccct cgaacttcac ctcggcgcgg gtcttgtagt tgccgtcgtc
      421 cttgaagaag atggtgcgct cctggacgta gccttcgggc atggcggact tgaagaagtc
      481 gtgctgcttc atgtggtcgg ggtagcggct gaagcactgc acgccgtggc tcagggtggt
      541 cacgagggtg ggccagggca cgggcagctt gccggtggtg cagatgaact tcagggtcag
      601 cttgccgtag gtggcatcgc cctcgccctc gccggacacg ctgaacttgt ggccgtttac
      661 gtcgccgtcc agctcgacca ggatgggcac caccccggtg aacagctcct cgcccttgct
      721 caccatggct ccctgaaaat acagattctc ggcggccgcc ttaaggctga gggtacccct
      781 ggggagagag gtcggtgatt cggtcaacga gggagccgac tgccgacgtg cgctccggag
      841 gcttgcagaa tgcggaacac cgcgcgggca ggaacagggc ccacactacc gccccacacc
      901 ccgcctcccg caccgcccct tcccggccgc tgctctcggc gcgccctgct gagcagccgc
      961 tattggccac agcccatcgc ggtcggcgcg ctgccattgc tccctggcgc tgtccgtctg
     1021 cgagggtact agtgagacgt gcggcttccg tttgtcacgt ccggcacgcc gcgaaccgca
     1081 aggaaccttc ccgacttagg ggcggagcag gaagcgtcgc cggggggccc acaagggtag
     1141 cggcgaagat ccgggtgacg ctgcgaacgg acgtgaagaa tgtgcgagac ccagggtcgg
     1201 cgccgctgcg tttcccggaa ccacgcccag agcagccgcg tccctgcgca aacccagggc
     1261 tgccttggaa aaggcgcaac cccaaccccg tggatgcatt aaaaaacctc ccacacctcc
     1321 ccctgaacct gaaacaagct tgtgaggtgt gagctgtgtc ttcattcact ggcactatgc
     1381 tcccagcatc catgaggctg gcgttgggaa aagaaattct caggaccagt atccagtgtg
     1441 tgtcccaatc ccggcttcac agaatcagta ttaccactct tcaaggcagc agttaacatt
     1501 tgtttatcag atgggaatct ttatttttgt atacttcacc taaaagattt ttaaatggga
     1561 gggcaagaat tttataacaa aaggttcaca atattagctt ccttattttt agtaactaaa
     1621 ttccttctcc ccaccccttc ccaggttcct gccatctccc tggcttatga gcaggctgag
     1681 agtgacatca tgaagagaca gcccagaaat cccaaaacag acaaacttgt gaatgagcgg
     1741 ctgatcagca tggcctatgg gcagattggt aagctgcagc ctggagtggg aagctggcac
     1801 atctaaggca tctgaggtga tggtgtccac ctcaggttga ggttgatttc agagactgca
     1861 aatccaggcg actttcaggt ctaggatgag ccctaacgga gtgagcctgt ggagtttctc
     1921 tgaactctct tccatgtgag aatacatctg ttgctgtttg cccagttacc ttttgtggaa
     1981 actggttttc tatactgagc accttggaac tgggctcaac tctgcctgag aaatgaggat
     2041 gtccttcctc actgagcctt gcggaacagg aggcatggtt ctaagggcta gccggaagcg
     2101 ctcctccctc tgcgagagca atggctccgt ccggtttcga gccatatggt gcactctcag
     2161 tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga
     2221 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     2281 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg
     2341 cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc
     2401 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     2461 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     2521 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     2581 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     2641 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     2701 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     2761 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     2821 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     2881 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     2941 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     3001 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     3061 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     3121 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     3181 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     3241 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     3301 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     3361 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     3421 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     3481 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     3541 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     3601 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     3661 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     3721 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     3781 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     3841 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     3901 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     3961 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     4021 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     4081 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     4141 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     4201 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     4261 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     4321 ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta
     4381 atgcagaatt ccacccacca gagtcattac tttgagtagc tcttgttaat gctgggggct
     4441 atgtttgttg tcacttctca gttctgttat ttggtgtagg gcctgtgtga atacttgcct
     4501 gtgacggttc tcaggcttca taaatagtct caataggaaa ggagcagtgt ctgtaatgag
     4561 tgctcagtgg gggcatgcat cgcactattt ccatcgctag gaaaagtgat tggtattaac
     4621 ccgttttccc ttttctgggg tagggtgcta tcgtggctgt gactggtgac ggtgtgaatg
     4681 actctccagc tttgaagaaa gcagacattg gggttgctat ggggattgct ggctcagatg
     4741 tgtccaagca agctgctgac atgattcttc tggatgacaa ctttgcctca attgtgactg
     4801 gagtagagga aggtgagagc tatttaaggt gtacaccaag atcttattca gatactgccc
     4861 attagcatcc atttctgtat acttcttgga tatgttcagt ttccagtgtg cttgtctcat
     4921 aagctaacag taaaaaatct tggttttcat aggtcgtctg atctttgata acttgaagaa
     4981 atccattgct tataccttaa ccagtaacat tcccgagatc accccgttcc tgatatttat
     5041 tattgcaaac attccactac cactggggaa tgtcaccatc ctctgcattg acttgggcac
     5101 tgacatggtg agtgtcacaa cagtcacaga tcgatatcag acgtgatatg tcgacccata
     5161 gagcccaccg catccccagc atgcctgcta ttgtcttccc aatcctcccc cttgctgtcc
     5221 tgccccaccc caccccccag aatagaatga cacctactca gacaatgcga tgcaatttcc
     5281 tcattttatt aggaaaggac agtgggagtg gcaccttcca gggtcaagga aggcacgggg
     5341 gaggggcaaa caacagatgg ctggcaacta gaaggcacag cc
//
