LOCUS       sequence-222264-        4037 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 4037)
  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 4037)
  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-cmlc2-eGFP
FEATURES             Location/Qualifiers
     source          1..4037
                     /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             2546..2566
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(2570..2572,2573..2575,2576..3289)
                     /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(2570..2572,2573..2575,2576..3286)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2615..2636)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2876..2895)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3223..3244
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3294..3518
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    3588..3669
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    3793..3874
                     /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 gcggcattca tccatccttt tcatccctca aatctctcat tcacgtcccc
     2041 ctccccatct gcacacttta tctcattttc caccctgctg gaatctgagc acttgtgcag
     2101 ttatcagggc tcctgtattt aggaggctct gggtgtccat gtaggggacg aacagaaaca
     2161 ctgcagacct ttatagaaga acaaatgata agagtcctca tacataaaga ctccattaga
     2221 aacgtcagtg acccaggagc ccagaccaac agcaaagcag acagtgaagg atccacagcc
     2281 tgactaactc gacaaacccc cccaaaccta aggtgagttg atctttaagc tttttacatt
     2341 ttcagctcgc atatatcaat tcgcgcgcat tgtcacacta gcacccacta gcggtcagac
     2401 tgcagattgc agcacgaaac aggaagctga ctccacatgg tcacatgctc actgaagtgt
     2461 tgacttccct gacagctgtg cactttctaa accggttttc tcattcattt acagttcagc
     2521 cggaattcac ctcgagacca tggctccaaa gaagaagcgt aaggtaacca tggtgagcaa
     2581 gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa
     2641 cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac
     2701 cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac
     2761 cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt
     2821 cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga
     2881 cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat
     2941 cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta
     3001 caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt
     3061 gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca
     3121 gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac
     3181 ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt
     3241 cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagtaaa gatctgtgcc
     3301 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     3361 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     3421 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     3481 caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccaa
     3541 agggcgaatt aattcattga tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa
     3601 tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat aagctgcaat
     3661 aaacaagtta acaacaacaa ttgcattcat tttatgtttc aggttcaggg ggaggtgtgg
     3721 gaggtttttt aaagcaagta aaacctctac aaatgtggtc attgatgagt ttggacaaac
     3781 cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt
     3841 atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca ttcattttat
     3901 gtttcaggtt cagggggagg tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg
     3961 tggtcattca gatcaattaa ccctcacccg ggctagcgat gcatgggagc tctgacgtac
     4021 gttgtctagc aaggaag
//
