LOCUS       sequence-227652-        6931 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 6931)
  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 6931)
  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-gal4-VP16-gcry-TagBFP
FEATURES             Location/Qualifiers
     source          1..6931
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     224..241
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    join(271..282,283..445)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     protein_bind    532..553
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(568..573,574..591,592..598)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    606..622
                     /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     611..633
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     630..646
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     630..646
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             642..998
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWTCHTSRSHELLS
                     VKASRRRGRKPRSF"
     promoter        667..685
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        complement(733..751)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(761..778)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(761..777)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(770..792)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             937..993
                     /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             1000..1437
                     /codon_start=1
                     /gene="Saccharomyces cerevisiae GAL4 (truncated)"
                     /product="DNA binding domain of the GAL4 transcriptional 
                     activator"
                     /label=GAL4 DNA binding domain
                     /translation="KLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTKR
                     SPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNKD
                     AVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVS"
     CDS             3524..3544
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3548..4249
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     polyA_signal    4259..4483
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    4553..4634
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    4758..4839
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        5156..5174
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(5197..5384,5385..5396)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(5450..5469)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5461..6270
                     /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(5528..5547)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      join(6413..6931,1..70)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6902..6921
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
       61 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
      121 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
      181 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
      241 ggaagcggaa cagatttaaa tggtaccgag cagaggtgta aaaagtactc aaaaatttta
      301 ctcaagtgaa agtacaagta cttagggaaa attttactca attaaaagta aaagtatctg
      361 gctagaatct tacttgagta aaagtaaaaa agtactccat taaaattgta cttgagtatt
      421 aaggaagtaa aagtaaaagc aagaaagaaa actagagtgg tctcccttta gtgagggtca
      481 attgatggga ggcgttcggg ccacagcgga cacgcaggtg cgcaacgcaa ttaatgtgag
      541 ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg
      601 tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa
      661 gcgcgcaatt aaccctcact aaagggaaca aaagctggag gacctcgagg gggggcccgg
      721 tacccaattc gccctatagt gagtcgtatt acgcgcgctc actggccgtc gttttacaac
      781 gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt
      841 tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca
      901 gcctgaatgg cgaatggacc tgccacactt cccggagcca cgaacttctc tctgttaaag
      961 caagcaggag acgtggaaga aaaccccggt ccttctagaa agctactgtc ttctatcgaa
     1021 caagcatgcg atatttgccg acttaaaaag ctcaagtgct ccaaagaaaa accgaagtgc
     1081 gccaagtgtc tgaagaacaa ctgggagtgt cgctactctc ccaaaaccaa aaggtctccg
     1141 ctgactaggg cacatctgac agaagtggaa tcaaggctag aaagactgga acagctattt
     1201 ctactgattt ttcctcgaga agaccttgac atgattttga aaatggattc tttacaggat
     1261 ataaaagcat tgttaacagg attatttgta caagataatg tgaataaaga tgccgtcaca
     1321 gatagattgg cttcagtgga gactgatatg cctctaacat tgagacagca tagaataagt
     1381 gcgacatcat catcggaaga gagtagtaac aaaggtcaaa gacagttgac tgtatcgtcg
     1441 aggtcgaccc cgggaataca gatctctcga gccgcccccc cgaccgatgt cagcctgggg
     1501 gacgagctcc acttagacgg cgaggacgtg gcgatggcgc atgccgacgc gctagacgat
     1561 ttcgatctgg acatgttggg ggacggggat tccccgggtc cgggatttac cccccacgac
     1621 tccgccccct acggcgctct ggatatgtaa aactagtaat taagtctcag ccaccgttaa
     1681 ctgaacatgt caaaacctgt ggagactgtt gagatttgat gttctgaaaa gataaagcct
     1741 ataaataaaa tgttgcccaa atttcctgcc tgatgttttt ctttgtcttt gctacatggc
     1801 tttgctgctc ggatcggctc actctgtgta tgccacgttc actttgtact ctccttctca
     1861 cggtaggttt attattttta gatgtgcagt tagtttctgt gaaataacac accacacact
     1921 gatattgtct gtgcattgac ttggtgagtg cacattgttt ttgatcttga catatttata
     1981 tttgattgat caggtgaact gtgtgaatct aaagtgctcc atacagatgt tctgcattga
     2041 aaatattctc attttattag tggaagtgag tgtatgccac atccaatcaa tttcagcaaa
     2101 caccccagta tgatttaatg caaaaaaatg aaggtatcaa acacgcatta ctactttgca
     2161 gttaaatatt taacatttat tccaacacga aaaaaagcag taaataacac tttgacaaac
     2221 acgtcaggac atcttatttt tgtcaccctc acaggcaatt tagtataata tattatatat
     2281 atatatatat catataataa tattcagtat aatatatata tatatatcat attataatat
     2341 tcagtataat ataaaacaca aacacatata tgtataatat aatataacat ttttatttat
     2401 tgagatgcct ctatggaccg tgttataaga agtaaagatc aggagaagta aacatgaagt
     2461 gtaattatga atactgatgt taaattaagc tatgatgagt tttcactgtt aatttaccat
     2521 ctcaattaaa tgttgatgcc tccatgacca agttaagcag atgagactga ggacaactgt
     2581 agaagacaag atgttcactt tgctgaatat agctggcttg acagttatct atgactctat
     2641 aaatatatat atattttttt tttataaaat gatttattta taactatata tccatttctc
     2701 agacaggtgc ttcatatccc tcactcccgt agctgtccat gctggatctg tccccgttgt
     2761 ttttaaaaag ctaaataagt tattaacatg actgcatcca gcgagccaaa cctgtctggt
     2821 gtacagctac cagagaagct tgagatccta gtcaccggcg ttcctagttc tttaaactgt
     2881 tacaaagtgt ttggaccagc aactgaattg cactgtaaat taccacatca gggtctccgg
     2941 ggtcaacgct gctaagcaat ggactggaaa atttgtgaca ttgagttttt ggaataactg
     3001 ggaatgctgg ataactcttt gggtaaagct atggaacaaa agtaccagca tgaactaaga
     3061 aatgactgtc acagcacaac atcaaacaca atgcaagaca aaggttctct gcttttgcct
     3121 tacttgacag aaaaagtaag ctctaggttc atcgcatgca gacagcagca atacatgaca
     3181 tatataaagg ctggctcttt tctcccttcc attgaactga aacttccact cagtcagact
     3241 tgcgggggat ccacagcctg actaactcga caaacccccc caaacctaag gtgagttgat
     3301 ctttaagctt tttacatttt cagctcgcat atatcaattc gcgcgcattg tcacactagc
     3361 acccactagc ggtcagactg cagattgcag cacgaaacag gaagctgact ccacatggtc
     3421 acatgctcac tgaagtgttg acttccctga cagctgtgca ctttctaaac cggttttctc
     3481 attcatttac agttcagccg gaattcacct cgagaccatg gctccaaaga agaagcgtaa
     3541 ggtaaccatg agcgagctga ttaaggagaa catgcacatg aagctgtaca tggagggcac
     3601 cgtggacaac catcacttca agtgcacatc cgagggcgaa ggcaagccct acgagggcac
     3661 ccagaccatg agaatcaagg tggtcgaggg cggccctctc cccttcgcct tcgacatcct
     3721 ggctactagc ttcctctacg gcagcaagac cttcatcaac cacacccagg gcatccccga
     3781 cttcttcaag cagtccttcc ctgagggctt cacatgggag agagtcacca catacgaaga
     3841 cgggggcgtg ctgaccgcta cccaggacac cagcctccag gacggctgcc tcatctacaa
     3901 cgtcaagatc agaggggtga acttcacatc caacggccct gtgatgcaga agaaaacact
     3961 cggctgggag gccttcaccg agacgctgta ccccgctgac ggcggcctgg aaggcagaaa
     4021 cgacatggcc ctgaagctcg tgggcgggag ccatctgatc gcaaacatca agaccacata
     4081 tagatccaag aaacccgcta agaacctcaa gatgcctggc gtctactatg tggactacag
     4141 actggaaaga atcaaggagg ccaacaacga gacctacgtc gagcagcacg aggtggcagt
     4201 ggccagatac tgcgacctcc ctagcaaact ggggcacaag ctcaattaaa gctgagatct
     4261 gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg
     4321 gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg
     4381 agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg
     4441 gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga
     4501 accaaagggc gaattaattc attgatgagt ttggacaaac cacaactaga atgcagtgaa
     4561 aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct
     4621 gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt cagggggagg
     4681 tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg tggtcattga tgagtttgga
     4741 caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt
     4801 gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa ttgcattcat
     4861 tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt aaagcaagta aaacctctac
     4921 aaatgtggtc attcagatca attaaccctc acccgggcta gcgatgcatg ggagctctga
     4981 cgtacgttgt ctagcaagga agtgaagagc tatccggata tatatgtatg ttatcataac
     5041 tgtactggct atgtcaggtg gtaatgactg taacgttacg ttactcgttg taggcacgac
     5101 attgaatatg ctcttctccg ctgtggcccg aacgcctccc gctagcgatg ggatctaata
     5161 cgactcacta tagggagacc aagggaaaat agaatgaagt gatctccaaa aaataagtac
     5221 tttttgactg taaataaaat tgtaaggagt aaaaagtact tttttttcta aaaaaatgta
     5281 attaagtaaa agtaaaagta ttgattttta attgtactca agtaaagtaa aaatccccaa
     5341 aaataatact taagtacagt aatcaagtaa aattactcaa gtactttaca cctctgggat
     5401 ccagctgcat catgaacaat aaaactgtct gcttacataa acagtaatac aaggggtgtt
     5461 atgagccata ttcaacggga aacgtcgagg ccgcgattaa attccaacat ggatgctgat
     5521 ttatatgggt ataaatgggc tcgcgataat gtcgggcaat caggtgcgac aatctatcgc
     5581 ttgtatggga agcccgatgc gccagagttg tttctgaaac atggcaaagg tagcgttgcc
     5641 aatgatgtta cagatgagat ggtcagacta aactggctga cggaatttat gcctcttccg
     5701 accatcaagc attttatccg tactcctgat gatgcatggt tactcaccac tgcgatcccc
     5761 ggaaaaacag cattccaggt attagaagaa tatcctgatt caggtgaaaa tattgttgat
     5821 gcgctggcag tgttcctgcg ccggttgcat tcgattcctg tttgtaattg tccttttaac
     5881 agcgatcgcg tatttcgtct cgctcaggcg caatcacgaa tgaataacgg tttggttgat
     5941 gcgagtgatt ttgatgacga gcgtaatggc tggcctgttg aacaagtctg gaaagaaatg
     6001 cataaacttt tgccattctc accggattca gtcgtcactc atggtgattt ctcacttgat
     6061 aaccttattt ttgacgaggg gaaattaata ggttgtattg atgttggacg agtcggaatc
     6121 gcagaccgat accaggatct tgccatccta tggaactgcc tcggtgagtt ttctccttca
     6181 ttacagaaac ggctttttca aaaatatggt attgataatc ctgatatgaa taaattgcag
     6241 tttcatttga tgctcgatga gtttttctaa tcagaattgg ttaattggtt gtaacactgg
     6301 caaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     6361 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     6421 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     6481 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     6541 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact
     6601 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     6661 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     6721 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     6781 aactgagata cctacagcgt gagctatgag aaagcgccac ggttcccgaa gggagaaagg
     6841 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     6901 ggggaaacgc ctggtatctt tatagtcctg t
//
