LOCUS       sequence-251957-        7282 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor for precise CRISPR directed genomic integration using the 
            GeneWeld method.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7282)
  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 7282)
  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..7282
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    join(14..25,26..188)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     protein_bind    275..296
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(311..316,317..334,335..341)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    349..365
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     354..376
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     373..389
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     373..389
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             385..798
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWTCHTSRSHELLS
                     VKASRRRGRKPRSYVQFTDRTPKFACITGRCNE"
     promoter        410..428
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        complement(476..494)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(504..521)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(504..520)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(513..535)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             680..736
                     /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             737..1768
                     /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
                     RYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLE
                     DGD"
     CDS             3642..3662
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3666..4343
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     CDS             3666..4343
                     /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(3796..3814)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4064..4083
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     4258..4281
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    4353..4577
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    4647..4728
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    4852..4933
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        5250..5268
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(5291..5478,5479..5490)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(5544..5563)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5555..6364
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(5622..5641)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      6507..7095
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6996..7015
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7249..7266
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 aaatggtacc gagcagaggt gtaaaaagta ctcaaaaatt ttactcaagt gaaagtacaa
       61 gtacttaggg aaaattttac tcaattaaaa gtaaaagtat ctggctagaa tcttacttga
      121 gtaaaagtaa aaaagtactc cattaaaatt gtacttgagt attaaggaag taaaagtaaa
      181 agcaagaaag aaaactagag tggtctccct ttagtgaggg tcaattgatg ggaggcgttc
      241 gggccacagc ggacacgcag gtgcgcaacg caattaatgt gagttagctc actcattagg
      301 caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat
      361 aacaatttca cacaggaaac agctatgacc atgattacgc caagcgcgca attaaccctc
      421 actaaaggga acaaaagctg gaggacctcg agggggggcc cggtacccaa ttcgccctat
      481 agtgagtcgt attacgcgcg ctcactggcc gtcgttttac aacgtcgtga ctgggaaaac
      541 cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat
      601 agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg
      661 acctgccaca cttcccggag ccacgaactt ctctctgtta aagcaagcag gagacgtgga
      721 agaaaacccc ggtcctatgt ccaatttact gaccgtacac caaaatttgc ctgcattacc
      781 ggtcgatgca acgagtgatg aggttcgcaa gaacctgatg gacatgttca gggatcgcca
      841 ggcgttttct gagcatacct ggaaaatgct tctgtccgtt tgccggtcgt gggcggcatg
      901 gtgcaagttg aataaccgga aatggtttcc cgcagaacct gaagatgttc gcgattatct
      961 tctatatctt caggcgcgcg gtctggcagt aaaaactatc cagcaacatt tgggccagct
     1021 aaacatgctt catcgtcggt ccgggctgcc acgaccaagt gacagcaatg ctgtttcact
     1081 ggttatgcgg cggatccgaa aagaaaacgt tgatgccggt gaacgtgcaa aacaggctct
     1141 agcgttcgaa cgcactgatt tcgaccaggt tcgttcactc atggaaaata gcgatcgctg
     1201 ccaggatata cgtaatctgg catttctggg gattgcttat aacaccctgt tacgtatagc
     1261 cgaaattgcc aggatcaggg ttaaagatat ctcacgtact gacggtggga gaatgttaat
     1321 ccatattggc agaacgaaaa cgctggttag caccgctgga gtagagaagg cacttagcct
     1381 gggggtaact aaactggtcg agcgatggat ttccgtctct ggtgtagctg atgatccgaa
     1441 taactacctg ttttgccggg tcagaaaaaa tggtgttgcc gcgccatctg ccaccagcca
     1501 gctatcaact cgcgccctgg aagggatttt tgaagcaact catcgattga tttacggcgc
     1561 taaggatgac tctggtcaga gatacctggc ctggtctgga cacagtgccc gtgtcggagc
     1621 cgcgcgagat atggcccgcg ctggagtttc aataccggag atcatgcaag ctggtggctg
     1681 gaccaatgta aatattgtca tgaactatat ccgtaacctg gatagtgaaa caggggcaat
     1741 ggtgcgcctg ctggaagatg gcgattagaa ctagtaatta agtctcagcc accgttaact
     1801 gaacatgtca aaacctgtgg agactgttga gatttgatgt tctgaaaaga taaagcctat
     1861 aaataaaatg ttgcccaaat ttcctgcctg atgtttttct ttgtctttgc tacatggctt
     1921 tgctgctcgg atcggctcac tctgtgtatg ccacgttcac tttgtactct ccttctcacg
     1981 gtaggtttat tatttttaga tgtgcagtta gtttctgtga aataacacac cacacactga
     2041 tattgtctgt gcattgactt ggtgagtgca cattgttttt gatcttgaca tatttatatt
     2101 tgattgatca ggtgaactgt gtgaatctaa agtgctccat acagatgttc tgcattgaaa
     2161 atattctcat tttattagtg gaagtgagtg tatgccacat ccaatcaatt tcagcaaaca
     2221 ccccagtatg atttaatgca aaaaaatgaa ggtatcaaac acgcattact actttgcagt
     2281 taaatattta acatttattc caacacgaaa aaaagcagta aataacactt tgacaaacac
     2341 gtcaggacat cttatttttg tcaccctcac aggcaattta gtataatata ttatatatat
     2401 atatatatca tataataata ttcagtataa tatatatata tatatcatat tataatattc
     2461 agtataatat aaaacacaaa cacatatatg tataatataa tataacattt ttatttattg
     2521 agatgcctct atggaccgtg ttataagaag taaagatcag gagaagtaaa catgaagtgt
     2581 aattatgaat actgatgtta aattaagcta tgatgagttt tcactgttaa tttaccatct
     2641 caattaaatg ttgatgcctc catgaccaag ttaagcagat gagactgagg acaactgtag
     2701 aagacaagat gttcactttg ctgaatatag ctggcttgac agttatctat gactctataa
     2761 atatatatat attttttttt tataaaatga tttatttata actatatatc catttctcag
     2821 acaggtgctt catatccctc actcccgtag ctgtccatgc tggatctgtc cccgttgttt
     2881 ttaaaaagct aaataagtta ttaacatgac tgcatccagc gagccaaacc tgtctggtgt
     2941 acagctacca gagaagcttg agatcctagt caccggcgtt cctagttctt taaactgtta
     3001 caaagtgttt ggaccagcaa ctgaattgca ctgtaaatta ccacatcagg gtctccgggg
     3061 tcaacgctgc taagcaatgg actggaaaat ttgtgacatt gagtttttgg aataactggg
     3121 aatgctggat aactctttgg gtaaagctat ggaacaaaag taccagcatg aactaagaaa
     3181 tgactgtcac agcacaacat caaacacaat gcaagacaaa ggttctctgc ttttgcctta
     3241 cttgacagaa aaagtaagct ctaggttcat cgcatgcaga cagcagcaat acatgacata
     3301 tataaaggct ggctcttttc tcccttccat tgaactgaaa cttccactca gtcagacttg
     3361 cgggggatcc acagcctgac taactcgaca aaccccccca aacctaaggt gagttgatct
     3421 ttaagctttt tacattttca gctcgcatat atcaattcgc gcgcattgtc acactagcac
     3481 ccactagcgg tcagactgca gattgcagca cgaaacagga agctgactcc acatggtcac
     3541 atgctcactg aagtgttgac ttccctgaca gctgtgcact ttctaaaccg gttttctcat
     3601 tcatttacag ttcagccgga attcacctcg agaccatggc tccaaagaag aagcgtaagg
     3661 taaccatggc ctcctccgag gacgtcatca aggagttcat gcgcttcaag gtgcgcatgg
     3721 agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc cgcccctacg
     3781 agggcaccca gaccgccaag ctgaaggtga ccaagggcgg ccccctgccc ttcgcctggg
     3841 acatcctgtc ccctcagttc cagtacggct ccaaggccta cgtgaagcac cccgccgaca
     3901 tccccgacta cttgaagctg tccttccccg agggcttcaa gtgggagcgc gtgatgaact
     3961 tcgaggacgg cggcgtggtg accgtgaccc aggactcctc cctgcaggac ggcgagttca
     4021 tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta atgcagaaga
     4081 agaccatggg ctgggaggcc tccaccgagc ggatgtaccc cgaggacggc gccctgaagg
     4141 gcgagatcaa gatgaggctg aagctgaagg acggcggcca ctacgacgcc gaggtcaaga
     4201 ccacctacat ggccaagaag cccgtgcagc tgcccggcgc ctacaagacc gacatcaagc
     4261 tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgag cgcgccgagg
     4321 gccgccactc caccggcgcc taaagctgtg atctgtgcct tctagttgcc agccatctgt
     4381 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc
     4441 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg
     4501 tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga
     4561 tgcggtgggc tctatggctt ctgaggcgga aagaaccaaa gggcgaatta attcattgat
     4621 gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt
     4681 gatgctattg ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat
     4741 tgcattcatt ttatgtttca ggttcagggg gaggtgtggg aggtttttta aagcaagtaa
     4801 aacctctaca aatgtggtca ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa
     4861 aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta tttgtaacca ttataagctg
     4921 caataaacaa gttaacaaca acaattgcat tcattttatg tttcaggttc agggggaggt
     4981 gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt ggtcattcag atcaattaac
     5041 cctcacccgg gctagcgatg catgggagct ctgacgtacg ttgtctagca aggaagtgaa
     5101 gagctatccg gatatatatg tatgttatca taactgtact ggctatgtca ggtggtaatg
     5161 actgtaacgt tacgttactc gttgtaggca cgacattgaa tatgctcttc tccgctgtgg
     5221 cccgaacgcc tcccgctagc gatgggatct aatacgactc actataggga gaccaaggga
     5281 aaatagaatg aagtgatctc caaaaaataa gtactttttg actgtaaata aaattgtaag
     5341 gagtaaaaag tacttttttt tctaaaaaaa tgtaattaag taaaagtaaa agtattgatt
     5401 tttaattgta ctcaagtaaa gtaaaaatcc ccaaaaataa tacttaagta cagtaatcaa
     5461 gtaaaattac tcaagtactt tacacctctg ggatccagct gcatcatgaa caataaaact
     5521 gtctgcttac ataaacagta atacaagggg tgttatgagc catattcaac gggaaacgtc
     5581 gaggccgcga ttaaattcca acatggatgc tgatttatat gggtataaat gggctcgcga
     5641 taatgtcggg caatcaggtg cgacaatcta tcgcttgtat gggaagcccg atgcgccaga
     5701 gttgtttctg aaacatggca aaggtagcgt tgccaatgat gttacagatg agatggtcag
     5761 actaaactgg ctgacggaat ttatgcctct tccgaccatc aagcatttta tccgtactcc
     5821 tgatgatgca tggttactca ccactgcgat ccccggaaaa acagcattcc aggtattaga
     5881 agaatatcct gattcaggtg aaaatattgt tgatgcgctg gcagtgttcc tgcgccggtt
     5941 gcattcgatt cctgtttgta attgtccttt taacagcgat cgcgtatttc gtctcgctca
     6001 ggcgcaatca cgaatgaata acggtttggt tgatgcgagt gattttgatg acgagcgtaa
     6061 tggctggcct gttgaacaag tctggaaaga aatgcataaa cttttgccat tctcaccgga
     6121 ttcagtcgtc actcatggtg atttctcact tgataacctt atttttgacg aggggaaatt
     6181 aataggttgt attgatgttg gacgagtcgg aatcgcagac cgataccagg atcttgccat
     6241 cctatggaac tgcctcggtg agttttctcc ttcattacag aaacggcttt ttcaaaaata
     6301 tggtattgat aatcctgata tgaataaatt gcagtttcat ttgatgctcg atgagttttt
     6361 ctaatcagaa ttggttaatt ggttgtaaca ctggcaaaaa ggatctaggt gaagatcctt
     6421 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     6481 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     6541 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     6601 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
     6661 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     6721 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     6781 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     6841 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     6901 tgagaaagcg ccacggttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     6961 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     7021 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     7081 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     7141 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     7201 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     7261 agcgaggaag cggaacagat tt
//
