LOCUS       sequence-222291-        4088 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 4088)
  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 4088)
  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-gcry-eGFP
FEATURES             Location/Qualifiers
     source          1..4088
                     /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             2597..2617
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(2621..2623,2624..2626,2627..3340)
                     /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(2621..2623,2624..2626,2627..3337)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2666..2687)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2927..2946)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3274..3295
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3345..3569
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    3639..3720
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    3844..3925
                     /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 gcgttcctag ttctttaaac tgttacaaag tgtttggacc agcaactgaa
     1981 ttgcactgta aattaccaca tcagggtctc cggggtcaac gctgctaagc aatggactgg
     2041 aaaatttgtg acattgagtt tttggaataa ctgggaatgc tggataactc tttgggtaaa
     2101 gctatggaac aaaagtacca gcatgaacta agaaatgact gtcacagcac aacatcaaac
     2161 acaatgcaag acaaaggttc tctgcttttg ccttacttga cagaaaaagt aagctctagg
     2221 ttcatcgcat gcagacagca gcaatacatg acatatataa aggctggctc ttttctccct
     2281 tccattgaac tgaaacttcc actcagtcag acttgcgggg gatccacagc ctgactaact
     2341 cgacaaaccc ccccaaacct aaggtgagtt gatctttaag ctttttacat tttcagctcg
     2401 catatatcaa ttcgcgcgca ttgtcacact agcacccact agcggtcaga ctgcagattg
     2461 cagcacgaaa caggaagctg actccacatg gtcacatgct cactgaagtg ttgacttccc
     2521 tgacagctgt gcactttcta aaccggtttt ctcattcatt tacagttcag ccggaattca
     2581 cctcgagacc atggctccaa agaagaagcg taaggtaacc atggtgagca agggcgagga
     2641 gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa
     2701 gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt
     2761 catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta
     2821 cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc
     2881 cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta
     2941 caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa
     3001 gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa
     3061 cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa
     3121 gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac
     3181 ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc
     3241 cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc
     3301 cgccgggatc actctcggca tggacgagct gtacaagtaa agatctgtgc cttctagttg
     3361 ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc
     3421 cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc
     3481 tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag
     3541 gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca aagggcgaat
     3601 taattcattg atgagtttgg acaaaccaca actagaatgc agtgaaaaaa atgctttatt
     3661 tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa taaacaagtt
     3721 aacaacaaca attgcattca ttttatgttt caggttcagg gggaggtgtg ggaggttttt
     3781 taaagcaagt aaaacctcta caaatgtggt cattgatgag tttggacaaa ccacaactag
     3841 aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac
     3901 cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta tgtttcaggt
     3961 tcagggggag gtgtgggagg ttttttaaag caagtaaaac ctctacaaat gtggtcattc
     4021 agatcaatta accctcaccc gggctagcga tgcatgggag ctctgacgta cgttgtctag
     4081 caaggaag
//
