LOCUS       sequence-222288-        4051 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 4051)
  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 4051)
  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-mRFP
FEATURES             Location/Qualifiers
     source          1..4051
                     /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             2621..3298
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     CDS             2621..3298
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /note="mammalian codon-optimized"
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     primer_bind     complement(2751..2769)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3019..3038
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     3213..3236
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    3308..3532
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    3602..3683
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    3807..3888
                     /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 atggcctcct ccgaggacgt
     2641 catcaaggag ttcatgcgct tcaaggtgcg catggagggc tccgtgaacg gccacgagtt
     2701 cgagatcgag ggcgagggcg agggccgccc ctacgagggc acccagaccg ccaagctgaa
     2761 ggtgaccaag ggcggccccc tgcccttcgc ctgggacatc ctgtcccctc agttccagta
     2821 cggctccaag gcctacgtga agcaccccgc cgacatcccc gactacttga agctgtcctt
     2881 ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag gacggcggcg tggtgaccgt
     2941 gacccaggac tcctccctgc aggacggcga gttcatctac aaggtgaagc tgcgcggcac
     3001 caacttcccc tccgacggcc ccgtaatgca gaagaagacc atgggctggg aggcctccac
     3061 cgagcggatg taccccgagg acggcgccct gaagggcgag atcaagatga ggctgaagct
     3121 gaaggacggc ggccactacg acgccgaggt caagaccacc tacatggcca agaagcccgt
     3181 gcagctgccc ggcgcctaca agaccgacat caagctggac atcacctccc acaacgagga
     3241 ctacaccatc gtggaacagt acgagcgcgc cgagggccgc cactccaccg gcgcctaaag
     3301 ctgagatctg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc
     3361 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg
     3421 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg
     3481 gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag
     3541 gcggaaagaa ccaaagggcg aattaattca ttgatgagtt tggacaaacc acaactagaa
     3601 tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta tttgtaacca
     3661 ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg tttcaggttc
     3721 agggggaggt gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt ggtcattgat
     3781 gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt
     3841 gatgctattg ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat
     3901 tgcattcatt ttatgtttca ggttcagggg gaggtgtggg aggtttttta aagcaagtaa
     3961 aacctctaca aatgtggtca ttcagatcaa ttaaccctca cccgggctag cgatgcatgg
     4021 gagctctgac gtacgttgtc tagcaaggaa g
//
