LOCUS       sequence-222300-        3977 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Donor for precise CRISPR directed genomic integration using the 
            GeneWeld method.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 3977)
  AUTHORS   Wierson WA, Welker JM, Almeida MP, Mann CM, Webster DA, Torrie ME, 
            Weiss TJ, Kambakam S, Vollbrecht MK, Lan M, McKeighan KC, Levey J, 
            Ming Z, Wehmeier A, Mikelson CS, Haltom JA, Kwan KM, Chien CB, 
            Balciunas D, Ekker SC, Clark KJ, Webber BR, Moriarity BS, Solin SL, 
            Carlson DF, Dobbs DL, McGrail M, Essner J
  TITLE     Efficient targeted integration directed by short homology in 
            zebrafish and mammalian cells.
  JOURNAL   Elife. 2020 May 15;9. pii: 53968. doi: 10.7554/eLife.53968.
  PUBMED    32412410
REFERENCE   2  (bases 1 to 3977)
  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: "Elife."; 
            date: "2020-05-15"; volume: "9. pii: 53968. doi"; pages: " 
            10.7554/eLife.53968"
COMMENT     Sequence Label: pPRISM-gal4-VP16-cmlc2-eGFP
FEATURES             Location/Qualifiers
     source          1..3977
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     CDS             10..66
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             73..510
                     /codon_start=1
                     /gene="Saccharomyces cerevisiae GAL4 (truncated)"
                     /product="DNA binding domain of the GAL4 transcriptional 
                     activator"
                     /label=GAL4 DNA binding domain
                     /translation="KLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTKR
                     SPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNKD
                     AVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVS"
     CDS             2486..2506
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(2510..2512,2513..2515,2516..3229)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2510..2512,2513..2515,2516..3226)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2555..2576)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2816..2835)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3163..3184
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3234..3458
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    3528..3609
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    3733..3814
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
ORIGIN
        1 cttcccggag ccacgaactt ctctctgtta aagcaagcag gagacgtgga agaaaacccc
       61 ggtccttcta gaaagctact gtcttctatc gaacaagcat gcgatatttg ccgacttaaa
      121 aagctcaagt gctccaaaga aaaaccgaag tgcgccaagt gtctgaagaa caactgggag
      181 tgtcgctact ctcccaaaac caaaaggtct ccgctgacta gggcacatct gacagaagtg
      241 gaatcaaggc tagaaagact ggaacagcta tttctactga tttttcctcg agaagacctt
      301 gacatgattt tgaaaatgga ttctttacag gatataaaag cattgttaac aggattattt
      361 gtacaagata atgtgaataa agatgccgtc acagatagat tggcttcagt ggagactgat
      421 atgcctctaa cattgagaca gcatagaata agtgcgacat catcatcgga agagagtagt
      481 aacaaaggtc aaagacagtt gactgtatcg tcgaggtcga ccccgggaat acagatctct
      541 cgagccgccc ccccgaccga tgtcagcctg ggggacgagc tccacttaga cggcgaggac
      601 gtggcgatgg cgcatgccga cgcgctagac gatttcgatc tggacatgtt gggggacggg
      661 gattccccgg gtccgggatt taccccccac gactccgccc cctacggcgc tctggatatg
      721 taaaactagt aattaagtct cagccaccgt taactgaaca tgtcaaaacc tgtggagact
      781 gttgagattt gatgttctga aaagataaag cctataaata aaatgttgcc caaatttcct
      841 gcctgatgtt tttctttgtc tttgctacat ggctttgctg ctcggatcgg ctcactctgt
      901 gtatgccacg ttcactttgt actctccttc tcacggtagg tttattattt ttagatgtgc
      961 agttagtttc tgtgaaataa cacaccacac actgatattg tctgtgcatt gacttggtga
     1021 gtgcacattg tttttgatct tgacatattt atatttgatt gatcaggtga actgtgtgaa
     1081 tctaaagtgc tccatacaga tgttctgcat tgaaaatatt ctcattttat tagtggaagt
     1141 gagtgtatgc cacatccaat caatttcagc aaacacccca gtatgattta atgcaaaaaa
     1201 atgaaggtat caaacacgca ttactacttt gcagttaaat atttaacatt tattccaaca
     1261 cgaaaaaaag cagtaaataa cactttgaca aacacgtcag gacatcttat ttttgtcacc
     1321 ctcacaggca atttagtata atatattata tatatatata tatcatataa taatattcag
     1381 tataatatat atatatatat catattataa tattcagtat aatataaaac acaaacacat
     1441 atatgtataa tataatataa catttttatt tattgagatg cctctatgga ccgtgttata
     1501 agaagtaaag atcaggagaa gtaaacatga agtgtaatta tgaatactga tgttaaatta
     1561 agctatgatg agttttcact gttaatttac catctcaatt aaatgttgat gcctccatga
     1621 ccaagttaag cagatgagac tgagacaact gtagaagaca agatgttcac tttgctgaat
     1681 atagctggct tgacagttat ctatgactct ataaatatat atatattttt ttttttataa
     1741 aatgatttat ttataactat atatccattt ctcagacagg tgcttcatat ccctcactcc
     1801 cgtagctgtc catgctggat ctgtccccgt tgtttttaaa aagctaaata agttattaac
     1861 atgactgcat ccagcgagcc aaacctgtct ggtgtacagc taccagagaa gcttgagatc
     1921 ctagtcaccg gcggcattca tccatccttt tcatccctca aatctctcat tcacgtcccc
     1981 ctccccatct gcacacttta tctcattttc caccctgctg gaatctgagc acttgtgcag
     2041 ttatcagggc tcctgtattt aggaggctct gggtgtccat gtaggggacg aacagaaaca
     2101 ctgcagacct ttatagaaga acaaatgata agagtcctca tacataaaga ctccattaga
     2161 aacgtcagtg acccaggagc ccagaccaac agcaaagcag acagtgaagg atccacagcc
     2221 tgactaactc gacaaacccc cccaaaccta aggtgagttg atctttaagc tttttacatt
     2281 ttcagctcgc atatatcaat tcgcgcgcat tgtcacacta gcacccacta gcggtcagac
     2341 tgcagattgc agcacgaaac aggaagctga ctccacatgg tcacatgctc actgaagtgt
     2401 tgacttccct gacagctgtg cactttctaa accggttttc tcattcattt acagttcagc
     2461 cggaattcac ctcgagacca tggctccaaa gaagaagcgt aaggtaacca tggtgagcaa
     2521 gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa
     2581 cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac
     2641 cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac
     2701 cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt
     2761 cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga
     2821 cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat
     2881 cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta
     2941 caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt
     3001 gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca
     3061 gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac
     3121 ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt
     3181 cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagtaaa gatctgtgcc
     3241 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     3301 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     3361 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     3421 caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccaa
     3481 agggcgaatt aattcattga tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa
     3541 tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat aagctgcaat
     3601 aaacaagtta acaacaacaa ttgcattcat tttatgtttc aggttcaggg ggaggtgtgg
     3661 gaggtttttt aaagcaagta aaacctctac aaatgtggtc attgatgagt ttggacaaac
     3721 cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt
     3781 atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca ttcattttat
     3841 gtttcaggtt cagggggagg tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg
     3901 tggtcattca gatcaattaa ccctcacccg ggctagcgat gcatgggagc tctgacgtac
     3961 gttgtctagc aaggaag
//
