LOCUS       sequence-222255-        4148 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 4148)
  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 4148)
  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-eGFP
FEATURES             Location/Qualifiers
     source          1..4148
                     /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             join(2681..2683,2684..2686,2687..3400)
                     /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(2681..2683,2684..2686,2687..3397)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2726..2747)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2987..3006)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3334..3355
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3405..3629
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    3699..3780
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    3904..3985
                     /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 atggtgagca agggcgagga
     2701 gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa
     2761 gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt
     2821 catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta
     2881 cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc
     2941 cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta
     3001 caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa
     3061 gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa
     3121 cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa
     3181 gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac
     3241 ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc
     3301 cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc
     3361 cgccgggatc actctcggca tggacgagct gtacaagtaa agatctgtgc cttctagttg
     3421 ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc
     3481 cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc
     3541 tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag
     3601 gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca aagggcgaat
     3661 taattcattg atgagtttgg acaaaccaca actagaatgc agtgaaaaaa atgctttatt
     3721 tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa taaacaagtt
     3781 aacaacaaca attgcattca ttttatgttt caggttcagg gggaggtgtg ggaggttttt
     3841 taaagcaagt aaaacctcta caaatgtggt cattgatgag tttggacaaa ccacaactag
     3901 aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac
     3961 cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta tgtttcaggt
     4021 tcagggggag gtgtgggagg ttttttaaag caagtaaaac ctctacaaat gtggtcattc
     4081 agatcaatta accctcaccc gggctagcga tgcatgggag ctctgacgta cgttgtctag
     4141 caaggaag
//
