LOCUS       sequence-222336-        4352 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 4352)
  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 4352)
  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-Cre-cmlc2-eGFP
FEATURES             Location/Qualifiers
     source          1..4352
                     /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             67..1098
                     /codon_start=1
                     /product="site-specific recombinase"
                     /label=Cre
                     /note="Cre is a site-specific recombinase from 
                     bacteriophage P1. Recombination occurs at loxP sequences."
                     /translation="MSNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKML
                     LSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGL
                     PRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAF
                     LGIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVE
                     RWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQ
                     RYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRTLDSETGAMVRLLE
                     DGD"
     CDS             2861..2881
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(2885..2887,2888..2890,2891..3604)
                     /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(2885..2887,2888..2890,2891..3601)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3191..3210)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3538..3559
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3609..3833
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    3903..3984
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    4108..4189
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
ORIGIN
        1 cttcccggag ccacgaactt ctctctgtta aagcaagcag gagacgtgga agaaaacccc
       61 ggtcctatgt ccaatttact gaccgtacac caaaatttgc ctgcattacc ggtcgatgca
      121 acgagtgatg aggttcgcaa gaacctgatg gacatgttca gggatcgcca ggcgttttct
      181 gagcatacct ggaaaatgct tctgtccgtt tgccggtcgt gggcggcatg gtgcaagttg
      241 aataaccgga aatggtttcc cgcagaacct gaagatgttc gcgattatct tctatatctt
      301 caggcgcgcg gtctggcagt aaaaactatc cagcaacatt tgggccagct aaacatgctt
      361 catcgtcggt ccgggctgcc acgaccaagt gacagcaatg ctgtttcact ggttatgcgg
      421 cggatccgaa aagaaaacgt tgatgccggt gaacgtgcaa aacaggctct agcgttcgaa
      481 cgcactgatt tcgaccaggt tcgttcactc atggaaaata gcgatcgctg ccaggatata
      541 cgtaatctgg catttctggg gattgcttat aacaccctgt tacgtatagc cgaaattgcc
      601 aggatcaggg ttaaagatat ctcacgtact gacggtggga gaatgttaat ccatattggc
      661 agaacgaaaa cgctggttag caccgctgga gtagagaagg cacttagcct gggggtaact
      721 aaactggtcg agcgatggat ttccgtctct ggtgtagctg atgatccgaa taactacctg
      781 ttttgccggg tcagaaaaaa tggtgttgcc gcgccatctg ccaccagcca gctatcaact
      841 cgcgccctgg aagggatttt tgaagcaact catcgattga tttacggcgc taaggatgac
      901 tctggtcaga gatacctggc ctggtctgga cacagtgccc gtgtcggagc cgcgcgagat
      961 atggcccgcg ctggagtttc aataccggag atcatgcaag ctggtggctg gaccaatgta
     1021 aatattgtca tgaactatat ccgtaccctg gatagtgaaa caggggcaat ggtgcgcctg
     1081 ctggaagatg gcgattagaa ctagtaatta agtctcagcc accgttaact gaacatgtca
     1141 aaacctgtgg agactgttga gatttgatgt tctgaaaaga taaagcctat aaataaaatg
     1201 ttgcccaaat ttcctgcctg atgtttttct ttgtctttgc tacatggctt tgctgctcgg
     1261 atcggctcac tctgtgtatg ccacgttcac tttgtactct ccttctcacg gtaggtttat
     1321 tatttttaga tgtgcagtta gtttctgtga aataacacac cacacactga tattgtctgt
     1381 gcattgactt ggtgagtgca cattgttttt gatcttgaca tatttatatt tgattgatca
     1441 ggtgaactgt gtgaatctaa agtgctccat acagatgttc tgcattgaaa atattctcat
     1501 tttattagtg gaagtgagtg tatgccacat ccaatcaatt tcagcaaaca ccccagtatg
     1561 atttaatgca aaaaaatgaa ggtatcaaac acgcattact actttgcagt taaatattta
     1621 acatttattc caacacgaaa aaaagcagta aataacactt tgacaaacac gtcaggacat
     1681 cttatttttg tcaccctcac aggcaattta gtataatata ttatatatat atatatatca
     1741 tataataata ttcagtataa tatatatata tatatcatat tataatattc agtataatat
     1801 aaaacacaaa cacatatatg tataatataa tataacattt ttatttattg agatgcctct
     1861 atggaccgtg ttataagaag taaagatcag gagaagtaaa catgaagtgt aattatgaat
     1921 actgatgtta aattaagcta tgatgagttt tcactgttaa tttaccatct caattaaatg
     1981 ttgatgcctc catgaccaag ttaagcagat gagactgaga caactgtaga agacaagatg
     2041 ttcactttgc tgaatatagc tggcttgaca gttatctatg actctataaa tatatatata
     2101 tttttttttt tataaaatga tttatttata actatatatc catttctcag acaggtgctt
     2161 catatccctc actcccgtag ctgtccatgc tggatctgtc cccgttgttt ttaaaaagct
     2221 aaataagtta ttaacatgac tgcatccagc gagccaaacc tgtctggtgt acagctacca
     2281 gagaagcttg agatcctagt caccggcggc attcatccat ccttttcatc cctcaaatct
     2341 ctcattcacg tccccctccc catctgcaca ctttatctca ttttccaccc tgctggaatc
     2401 tgagcacttg tgcagttatc agggctcctg tatttaggag gctctgggtg tccatgtagg
     2461 ggacgaacag aaacactgca gacctttata gaagaacaaa tgataagagt cctcatacat
     2521 aaagactcca ttagaaacgt cagtgaccca ggagcccaga ccaacagcaa agcagacagt
     2581 gaaggatcca cagcctgact aactcgacaa acccccccaa acctaaggtg agttgatctt
     2641 taagcttttt acattttcag ctcgcatata tcaattcgcg cgcattgtca cactagcacc
     2701 cactagcggt cagactgcag attgcagcac gaaacaggaa gctgactcca catggtcaca
     2761 tgctcactga agtgttgact tccctgacag ctgtgcactt tctaaaccgg ttttctcatt
     2821 catttacagt tcagccggaa ttcacctcga gaccatggct ccaaagaaga agcgtaaggt
     2881 aaccatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct
     2941 ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac
     3001 ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc
     3061 caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat
     3121 gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat
     3181 cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac
     3241 cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg
     3301 gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa
     3361 gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct
     3421 cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa
     3481 ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat
     3541 ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa
     3601 gtaaagatct gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
     3661 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
     3721 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
     3781 ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga
     3841 ggcggaaaga accaaagggc gaattaattc attgatgagt ttggacaaac cacaactaga
     3901 atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc
     3961 attataagct gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt
     4021 cagggggagg tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg tggtcattga
     4081 tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg
     4141 tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa
     4201 ttgcattcat tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt aaagcaagta
     4261 aaacctctac aaatgtggtc attcagatca attaaccctc acccgggcta gcgatgcatg
     4321 ggagctctga cgtacgttgt ctagcaagga ag
//
