LOCUS       sequence-222252-        4111 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 4111)
  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 4111)
  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-TagRFP-gcry-mRFP
FEATURES             Location/Qualifiers
     source          1..4111
                     /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..783
                     /codon_start=1
                     /product="monomeric derivative of red fluorescent protein 
                     from Entacmaea quadricolor (Merzlyak et al., 2007)"
                     /label=TagRFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMR
                     IKVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGV
                     LTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRSDM
                     ALKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAVA
                     RYCDLPSKLGHKLN"
     CDS             2657..2677
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2681..3358
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     CDS             2681..3358
                     /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(2811..2829)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3079..3098
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     3273..3296
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    3368..3592
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    3662..3743
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    3867..3948
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
ORIGIN
        1 cttcccggag ccacgaactt ctctctgtta aagcaagcag gagacgtgga agaaaacccc
       61 ggtcctctcg aggtgtctaa gggcgaggaa ctgattaagg agaacatgca catgaagctg
      121 tacatggagg gcaccgtgaa caaccaccac ttcaagtgca catccgaggg cgaaggcaag
      181 ccctacgagg gcacccagac catgagaatc aaggtggtcg agggcggccc tctccccttc
      241 gccttcgaca tcctggctac cagcttcatg tacggcagca gaaccttcat caaccacacc
      301 cagggcatcc ccgacttctt taagcagtcc ttccctgagg gcttcacatg ggagagagtc
      361 accacatacg aagacggggg cgtgctgacc gctacccagg acaccagcct ccaggacggc
      421 tgcctcatct acaacgtcaa gatcagaggg gtgaacttcc catccaacgg ccctgtgatg
      481 cagaagaaaa cactcggctg ggaggccaac accgagatgc tgtaccccgc tgacggcggc
      541 ctggaaggca gaagcgacat ggccctgaag ctcgtgggcg ggggccacct gatctgcaac
      601 ttcaagacca catacagatc caagaaaccc gctaagaacc tcaagatgcc cggcgtctac
      661 tatgtggacc acagactgga aagaatcaag gaggccgaca aagagaccta cgtcgagcag
      721 cacgaggtgg ctgtggccag atactgcgac ctccctagca aactggggca caaacttaat
      781 tgaaactagt aattaagtct cagccaccgt taactgaaca tgtcaaaacc tgtggagact
      841 gttgagattt gatgttctga aaagataaag cctataaata aaatgttgcc caaatttcct
      901 gcctgatgtt tttctttgtc tttgctacat ggctttgctg ctcggatcgg ctcactctgt
      961 gtatgccacg ttcactttgt actctccttc tcacggtagg tttattattt ttagatgtgc
     1021 agttagtttc tgtgaaataa cacaccacac actgatattg tctgtgcatt gacttggtga
     1081 gtgcacattg tttttgatct tgacatattt atatttgatt gatcaggtga actgtgtgaa
     1141 tctaaagtgc tccatacaga tgttctgcat tgaaaatatt ctcattttat tagtggaagt
     1201 gagtgtatgc cacatccaat caatttcagc aaacacccca gtatgattta atgcaaaaaa
     1261 atgaaggtat caaacacgca ttactacttt gcagttaaat atttaacatt tattccaaca
     1321 cgaaaaaaag cagtaaataa cactttgaca aacacgtcag gacatcttat ttttgtcacc
     1381 ctcacaggca atttagtata atatattata tatatatata tatcatataa taatattcag
     1441 tataatatat atatatatat catattataa tattcagtat aatataaaac acaaacacat
     1501 atatgtataa tataatataa catttttatt tattgagatg cctctatgga ccgtgttata
     1561 agaagtaaag atcaggagaa gtaaacatga agtgtaatta tgaatactga tgttaaatta
     1621 agctatgatg agttttcact gttaatttac catctcaatt aaatgttgat gcctccatga
     1681 ccaagttaag cagatgagac tgagacaact gtagaagaca agatgttcac tttgctgaat
     1741 atagctggct tgacagttat ctatgactct ataaatatat atatattttt ttttttataa
     1801 aatgatttat ttataactat atatccattt ctcagacagg tgcttcatat ccctcactcc
     1861 cgtagctgtc catgctggat ctgtccccgt tgtttttaaa aagctaaata agttattaac
     1921 atgactgcat ccagcgagcc aaacctgtct ggtgtacagc taccagagaa gcttgagatc
     1981 ctagtcaccg gcgttcctag ttctttaaac tgttacaaag tgtttggacc agcaactgaa
     2041 ttgcactgta aattaccaca tcagggtctc cggggtcaac gctgctaagc aatggactgg
     2101 aaaatttgtg acattgagtt tttggaataa ctgggaatgc tggataactc tttgggtaaa
     2161 gctatggaac aaaagtacca gcatgaacta agaaatgact gtcacagcac aacatcaaac
     2221 acaatgcaag acaaaggttc tctgcttttg ccttacttga cagaaaaagt aagctctagg
     2281 ttcatcgcat gcagacagca gcaatacatg acatatataa aggctggctc ttttctccct
     2341 tccattgaac tgaaacttcc actcagtcag acttgcgggg gatccacagc ctgactaact
     2401 cgacaaaccc ccccaaacct aaggtgagtt gatctttaag ctttttacat tttcagctcg
     2461 catatatcaa ttcgcgcgca ttgtcacact agcacccact agcggtcaga ctgcagattg
     2521 cagcacgaaa caggaagctg actccacatg gtcacatgct cactgaagtg ttgacttccc
     2581 tgacagctgt gcactttcta aaccggtttt ctcattcatt tacagttcag ccggaattca
     2641 cctcgagacc atggctccaa agaagaagcg taaggtaacc atggcctcct ccgaggacgt
     2701 catcaaggag ttcatgcgct tcaaggtgcg catggagggc tccgtgaacg gccacgagtt
     2761 cgagatcgag ggcgagggcg agggccgccc ctacgagggc acccagaccg ccaagctgaa
     2821 ggtgaccaag ggcggccccc tgcccttcgc ctgggacatc ctgtcccctc agttccagta
     2881 cggctccaag gcctacgtga agcaccccgc cgacatcccc gactacttga agctgtcctt
     2941 ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag gacggcggcg tggtgaccgt
     3001 gacccaggac tcctccctgc aggacggcga gttcatctac aaggtgaagc tgcgcggcac
     3061 caacttcccc tccgacggcc ccgtaatgca gaagaagacc atgggctggg aggcctccac
     3121 cgagcggatg taccccgagg acggcgccct gaagggcgag atcaagatga ggctgaagct
     3181 gaaggacggc ggccactacg acgccgaggt caagaccacc tacatggcca agaagcccgt
     3241 gcagctgccc ggcgcctaca agaccgacat caagctggac atcacctccc acaacgagga
     3301 ctacaccatc gtggaacagt acgagcgcgc cgagggccgc cactccaccg gcgcctaaag
     3361 ctgagatctg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc
     3421 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg
     3481 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg
     3541 gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag
     3601 gcggaaagaa ccaaagggcg aattaattca ttgatgagtt tggacaaacc acaactagaa
     3661 tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta tttgtaacca
     3721 ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg tttcaggttc
     3781 agggggaggt gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt ggtcattgat
     3841 gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt
     3901 gatgctattg ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat
     3961 tgcattcatt ttatgtttca ggttcagggg gaggtgtggg aggtttttta aagcaagtaa
     4021 aacctctaca aatgtggtca ttcagatcaa ttaaccctca cccgggctag cgatgcatgg
     4081 gagctctgac gtacgttgtc tagcaaggaa g
//
