LOCUS       sequence-350127-        4017 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  HDR donor plasmid to tag LMNA with mScarlet-I using 
            eSpCas9(1.1)_No_FLAG_LMNA_G2..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4017)
  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 4017)
  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: LMNA_mScarlet-I_Donor
FEATURES             Location/Qualifiers
     source          1..4017
                     /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"
     CDS             647..1342
                     /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(780..798)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1048..1067
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(2027..2044)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2198..2786)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2278..2297)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2957..3748,3749..3817))
                     /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     3580..3599
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3818..3922)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3990..4008
                     /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 accggcaagc ttggagccga cagtgctgag caggcaggag ccaagagagg
      241 ggaagcttga gcctcacgca gttaggggtg cgctggagag ggtggggccc gactccgcca
      301 caccccaacg gtccttcccc ctcctcacca ctcccgcccc cacccccaat ggatctggga
      361 ctgccccttt aagagtagtg gcccctcctc ccttcagagg aggacctatt agagcctttg
      421 ccccggcgtc ggtgactcag tgttcgcggg agcgccgcac ctacaccagc caacccagat
      481 cccgaggtcc gacagcgccc ggcccagatc cccacgcctg ccaggagcaa gccgagagcc
      541 agccggccgg cgcactccga ctccgagcag tctctgtcct tcgacccgag ccccgcgccc
      601 tttccgggac ccctgccccg cgggcagcgc tgccaacctg ccggccatgg tgagcaaggg
      661 cgaggcagtg atcaaggagt tcatgcggtt caaggtgcac atggagggct ccatgaacgg
      721 ccacgagttc gagatcgagg gcgagggcga gggccgcccc tacgagggca cccagaccgc
      781 caagctgaag gtgaccaagg gtggccccct gcccttctcc tgggacatcc tgtcccctca
      841 gttcatgtac ggctccaggg ccttcatcaa gcaccccgcc gacatccccg actactataa
      901 gcagtccttc cccgagggct tcaagtggga gcgcgtgatg aacttcgagg acggcggcgc
      961 cgtgaccgtg acccaggaca cctccctgga ggacggcacc ctgatctaca aggtgaagct
     1021 ccgcggcacc aacttccctc ctgacggccc cgtaatgcag aagaagacaa tgggctggga
     1081 agcatccacc gagcggttgt accccgagga cggcgtgctg aagggcgaca ttaagatggc
     1141 cctgcgcctg aaggacggcg gccgctacct ggcggacttc aagaccacct acaaggccaa
     1201 gaagcccgtg cagatgcccg gcgcctacaa cgtcgaccgc aagttggaca tcacctccca
     1261 caacgaggac tacaccgtgg tggaacagta cgaacgctcc gagggccgcc actccaccgg
     1321 cggcatggac gagctgtaca agaccccgtc ccagcggcgc gccacccgca gcggggcgca
     1381 ggccagctcc actccgctgt cgcccacccg catcacccgg ctgcaggaga aggaggacct
     1441 gcaggagctc aatgatcgct tggcggtcta catcgaccgt gtgcgctcgc tggaaacgga
     1501 gaacgcaggg ctgcgccttc gcatcaccga gtctgaagag gtggtcagcc gcgaggtgtc
     1561 cggcatcaag gccgcctacg aggccgagct cggggatgcc cgcaagaccc ttgactcagt
     1621 agccaaggag cgcgcccgcc tgcagctgga gctgagcaaa gtgcgtgagg agtttaagga
     1681 gctgaaagcg cggtgagttc gcccaggtgg ctgcgtgcct ggcggggagt ggagagggcg
     1741 gcgggccggc gcccctggcc ggccgcagga agggagtgag agggcctgga ggccgataac
     1801 tttgccatag tctcctccct ccccggaact gcccccagcg ggtgactggc agtgtcaagg
     1861 ggaattgtca agacaggaca gagagggaag tggtggtctc tgggagaggg tcggggagga
     1921 tataaggaat ggtgggggta tcagggacaa gttggcgaat tctgcattaa tgaatcggcc
     1981 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     2041 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     2101 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     2161 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     2221 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     2281 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     2341 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     2401 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     2461 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     2521 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     2581 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     2641 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     2701 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     2761 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     2821 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     2881 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     2941 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     3001 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     3061 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     3121 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     3181 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     3241 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     3301 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     3361 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     3421 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     3481 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     3541 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     3601 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     3661 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     3721 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     3781 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     3841 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     3901 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa
     3961 ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtc
//
