LOCUS       sequence-350129-        4531 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  HDR donor plasmid to tag NPM1 with mNeonGreen using 
            eSpCas9(1.1)_No_FLAG_NPM1_G6..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4531)
  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 4531)
  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: NPM1_mNeonGreen_Donor
FEATURES             Location/Qualifiers
     source          1..4531
                     /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             1037..1744
                     /codon_start=1
                     /product="bright monomeric yellow-green fluorescent protein
                     derived from LanYFP (Shaner et al., 2013)"
                     /label=mNeonGreen
                     /translation="MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYE
                     ELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFED
                     GASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIIS
                     TFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQ
                     KAFTDVMGMDELYK"
     primer_bind     complement(2541..2558)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2712..3300)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2792..2811)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3471..4262,4263..4331))
                     /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     4094..4113
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4332..4436)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4504..4522
                     /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 accctctctt agggcgatgt ccttgctaat ttggagactg attcagtccc
      241 cttttggccc ccaagttacg taaagataag gactttggag atgttttctc aggaaggaca
      301 gagctgaaaa acaaagttcc gtaagtctgg ccggtaacta catctttcct tcctaacaaa
      361 gaataagccg caattcactc tctctggcac ctgaactttg gggtaacgat taactgcgat
      421 ccgaagcctt caggcaagta aaaaattcct gaagtgatta cgcctgtttg gaggcttgca
      481 gggcactagg ggatggggaa aggtgaatcg aggtgctctc tggctcattc gcagccggct
      541 aacccgccat atcttacaca gctaagggct gccgacgcca ttttgcaggg tgggctgcgc
      601 agactcttgg cgggaggccg gcgcgcttga gcgggagagg acgtggaagg gttggaggtg
      661 gggagtgcgc gttacgactg gaaagcacgc gtgcgcacag gcgacagcag cggaggggtg
      721 gggccagtgt acgagtgcgc gtgctcggtg ggagcccgcg gagtacctgg aaggaggtgg
      781 gggcgaggta gaaaggagtg gggttgaaaa gcgcttgcgc aggacggcta cggtacgggg
      841 gtgggagggc ttcggagcac gcgcgcggag gcgggacttg ggaagcgctc gcgagatctt
      901 cagggtctat atataagcgc ggctagcctg cgtacgttcc ctggtgtgat tccgtcctgc
      961 gcggttgttc tctggagcag cgttctttta tctccgtccg ccttctctcc tacctaagtg
     1021 cgtgccgcca cccgccatgg tgagcaaggg cgaggaggat aacatggcct ctctcccagc
     1081 gacacatgag ttacacatct ttggctccat caacggtgtg gactttgaca tggtgggtca
     1141 gggcaccggc aatccaaatg atggttatga ggagttaaac ctgaagtcca ccaagggtga
     1201 cctccagttc tccccctgga ttctggtccc tcatatcggg tatggcttcc atcagtacct
     1261 gccctaccct gacgggatgt cgcctttcca ggccgctatg gtagatggct ccggatacca
     1321 agtccatcgc acaatgcagt ttgaagatgg tgcctccctt actgttaact accgctacac
     1381 ctacgaggga agccacatca aaggagaggc ccaggtgaag gggactggtt tccctgctga
     1441 cggtcctgtg atgaccaact cgctgaccgc tgcggactgg tgcaggtcga agaagactta
     1501 ccccaacgac aaaaccatca tcagtacctt taagtggagt tacaccactg gaaatggcaa
     1561 gcgctaccgg agcactgcgc ggaccaccta cacctttgcc aagccaatgg cggctaacta
     1621 tctgaagaac cagccgatgt acgtgttccg taagacggag ctcaagcact ccaagaccga
     1681 gctcaacttc aaggagtggc aaaaggcctt taccgatgtg atgggcatgg acgagctgta
     1741 caaggaagat tcgatggaca tggacatgag ccccctgagg ccccagaact atcttttcgg
     1801 taactgctgg ggggagctgg agcgaggccg agcggggcct ggtggcggtg agggtggggg
     1861 tgaggggcgg gaatccggct gcaaccgggt ctggtggaga ccgccagacc gactggaggc
     1921 ctgagcgggt gggaagagct gcctgagccc tggacctgga gggattggag ccgcgggggg
     1981 agccggtggc gtgaaggggg aggagacgag cggcctgaag cgtctggggc gtgagcggtc
     2041 cgaggcccgg ggcctgaggt aaagtggaac cggccgcctg gaagttcggc ctttgccggg
     2101 gatacgtggc ggagctccag gcgtggttta ttttctaacg gcggtggttc ataggcttct
     2161 gatgccaggc cccgggttgg tggtcctgga ggctgggttc accgggaagc atgggcctgc
     2221 ttgttggagc gggtagatgc acagcctggc gttattctgg cctcagagcc tggagctgcc
     2281 ggacgtagac ttacctcgcg tgcttcggcc agttactggg ggtggggagg cgcggcgcgg
     2341 cgtgaggagg ccgcaaccgc tgggagcacg tggttgccac gtggttgggg gagggagggg
     2401 ggtgtgggcg ctacatccgg gactcaccgg cgtgttgcca ctcgtgagcc agggatgctg
     2461 tggctctgtt gaattctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     2521 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     2581 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     2641 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     2701 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     2761 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     2821 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     2881 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     2941 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     3001 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     3061 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     3121 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     3181 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     3241 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     3301 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     3361 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     3421 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     3481 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     3541 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     3601 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     3661 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     3721 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     3781 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     3841 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     3901 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     3961 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     4021 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     4081 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     4141 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     4201 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     4261 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     4321 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     4381 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     4441 tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat
     4501 aaaaataggc gtatcacgag gccctttcgt c
//
