LOCUS       sequence-222297-        3940 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 3940)
  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 3940)
  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-mRFP
FEATURES             Location/Qualifiers
     source          1..3940
                     /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             2510..3187
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     CDS             2510..3187
                     /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(2640..2658)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2908..2927
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     3102..3125
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    3197..3421
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    3491..3572
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    3696..3777
                     /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 tggcctcctc
     2521 cgaggacgtc atcaaggagt tcatgcgctt caaggtgcgc atggagggct ccgtgaacgg
     2581 ccacgagttc gagatcgagg gcgagggcga gggccgcccc tacgagggca cccagaccgc
     2641 caagctgaag gtgaccaagg gcggccccct gcccttcgcc tgggacatcc tgtcccctca
     2701 gttccagtac ggctccaagg cctacgtgaa gcaccccgcc gacatccccg actacttgaa
     2761 gctgtccttc cccgagggct tcaagtggga gcgcgtgatg aacttcgagg acggcggcgt
     2821 ggtgaccgtg acccaggact cctccctgca ggacggcgag ttcatctaca aggtgaagct
     2881 gcgcggcacc aacttcccct ccgacggccc cgtaatgcag aagaagacca tgggctggga
     2941 ggcctccacc gagcggatgt accccgagga cggcgccctg aagggcgaga tcaagatgag
     3001 gctgaagctg aaggacggcg gccactacga cgccgaggtc aagaccacct acatggccaa
     3061 gaagcccgtg cagctgcccg gcgcctacaa gaccgacatc aagctggaca tcacctccca
     3121 caacgaggac tacaccatcg tggaacagta cgagcgcgcc gagggccgcc actccaccgg
     3181 cgcctaaagc tgagatctgt gccttctagt tgccagccat ctgttgtttg cccctccccc
     3241 gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa
     3301 attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac
     3361 agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg
     3421 gcttctgagg cggaaagaac caaagggcga attaattcat tgatgagttt ggacaaacca
     3481 caactagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat
     3541 ttgtaaccat tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt
     3601 ttcaggttca gggggaggtg tgggaggttt tttaaagcaa gtaaaacctc tacaaatgtg
     3661 gtcattgatg agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt
     3721 gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa acaagttaac
     3781 aacaacaatt gcattcattt tatgtttcag gttcaggggg aggtgtggga ggttttttaa
     3841 agcaagtaaa acctctacaa atgtggtcat tcagatcaat taaccctcac ccgggctagc
     3901 gatgcatggg agctctgacg tacgttgtct agcaaggaag
//
