LOCUS       sequence-222327-        4463 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 4463)
  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 4463)
  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-gcry-eGFP
FEATURES             Location/Qualifiers
     source          1..4463
                     /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             2972..2992
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(2996..2998,2999..3001,3002..3715)
                     /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(2996..2998,2999..3001,3002..3712)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3302..3321)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3649..3670
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3720..3944
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    4014..4095
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    4219..4300
                     /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 caccggcgtt cctagttctt taaactgtta caaagtgttt
     2341 ggaccagcaa ctgaattgca ctgtaaatta ccacatcagg gtctccgggg tcaacgctgc
     2401 taagcaatgg actggaaaat ttgtgacatt gagtttttgg aataactggg aatgctggat
     2461 aactctttgg gtaaagctat ggaacaaaag taccagcatg aactaagaaa tgactgtcac
     2521 agcacaacat caaacacaat gcaagacaaa ggttctctgc ttttgcctta cttgacagaa
     2581 aaagtaagct ctaggttcat cgcatgcaga cagcagcaat acatgacata tataaaggct
     2641 ggctcttttc tcccttccat tgaactgaaa cttccactca gtcagacttg cgggggatcc
     2701 acagcctgac taactcgaca aaccccccca aacctaaggt gagttgatct ttaagctttt
     2761 tacattttca gctcgcatat atcaattcgc gcgcattgtc acactagcac ccactagcgg
     2821 tcagactgca gattgcagca cgaaacagga agctgactcc acatggtcac atgctcactg
     2881 aagtgttgac ttccctgaca gctgtgcact ttctaaaccg gttttctcat tcatttacag
     2941 ttcagccgga attcacctcg agaccatggc tccaaagaag aagcgtaagg taaccatggt
     3001 gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga
     3061 cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa
     3121 gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt
     3181 gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca
     3241 cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa
     3301 ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa
     3361 ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct
     3421 ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat
     3481 caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca
     3541 ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct
     3601 gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct
     3661 ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca agtaaagatc
     3721 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
     3781 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
     3841 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
     3901 ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag
     3961 aaccaaaggg cgaattaatt cattgatgag tttggacaaa ccacaactag aatgcagtga
     4021 aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc
     4081 tgcaataaac aagttaacaa caacaattgc attcatttta tgtttcaggt tcagggggag
     4141 gtgtgggagg ttttttaaag caagtaaaac ctctacaaat gtggtcattg atgagtttgg
     4201 acaaaccaca actagaatgc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat
     4261 tgctttattt gtaaccatta taagctgcaa taaacaagtt aacaacaaca attgcattca
     4321 ttttatgttt caggttcagg gggaggtgtg ggaggttttt taaagcaagt aaaacctcta
     4381 caaatgtggt cattcagatc aattaaccct cacccgggct agcgatgcat gggagctctg
     4441 acgtacgttg tctagcaagg aag
//
