LOCUS       sequence-222324-        4426 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 4426)
  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 4426)
  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-mRFP
FEATURES             Location/Qualifiers
     source          1..4426
                     /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             2996..3673
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     CDS             2996..3673
                     /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(3126..3144)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3394..3413
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     3588..3611
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    3683..3907
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    3977..4058
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    4182..4263
                     /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 taaccatggc
     3001 ctcctccgag gacgtcatca aggagttcat gcgcttcaag gtgcgcatgg agggctccgt
     3061 gaacggccac gagttcgaga tcgagggcga gggcgagggc cgcccctacg agggcaccca
     3121 gaccgccaag ctgaaggtga ccaagggcgg ccccctgccc ttcgcctggg acatcctgtc
     3181 ccctcagttc cagtacggct ccaaggccta cgtgaagcac cccgccgaca tccccgacta
     3241 cttgaagctg tccttccccg agggcttcaa gtgggagcgc gtgatgaact tcgaggacgg
     3301 cggcgtggtg accgtgaccc aggactcctc cctgcaggac ggcgagttca tctacaaggt
     3361 gaagctgcgc ggcaccaact tcccctccga cggccccgta atgcagaaga agaccatggg
     3421 ctgggaggcc tccaccgagc ggatgtaccc cgaggacggc gccctgaagg gcgagatcaa
     3481 gatgaggctg aagctgaagg acggcggcca ctacgacgcc gaggtcaaga ccacctacat
     3541 ggccaagaag cccgtgcagc tgcccggcgc ctacaagacc gacatcaagc tggacatcac
     3601 ctcccacaac gaggactaca ccatcgtgga acagtacgag cgcgccgagg gccgccactc
     3661 caccggcgcc taaagctgag atctgtgcct tctagttgcc agccatctgt tgtttgcccc
     3721 tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat
     3781 gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg
     3841 caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc
     3901 tctatggctt ctgaggcgga aagaaccaaa gggcgaatta attcattgat gagtttggac
     3961 aaaccacaac tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg
     4021 ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt
     4081 ttatgtttca ggttcagggg gaggtgtggg aggtttttta aagcaagtaa aacctctaca
     4141 aatgtggtca ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt
     4201 atttgtgaaa tttgtgatgc tattgcttta tttgtaacca ttataagctg caataaacaa
     4261 gttaacaaca acaattgcat tcattttatg tttcaggttc agggggaggt gtgggaggtt
     4321 ttttaaagca agtaaaacct ctacaaatgt ggtcattcag atcaattaac cctcacccgg
     4381 gctagcgatg catgggagct ctgacgtacg ttgtctagca aggaag
//
