LOCUS       sequence-229907-        6906 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 6906)
  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 6906)
  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-mRFP
FEATURES             Location/Qualifiers
     source          1..6906
                     /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..741
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWTCHTSRSHELLS
                     VKASRRRGRKPRSF"
     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             743..1180
                     /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             3267..3287
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3291..3968
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     CDS             3291..3968
                     /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(3421..3439)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3689..3708
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     3883..3906
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    3978..4202
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    4272..4353
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    4477..4558
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        4875..4893
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(4916..5103,5104..5115)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(5169..5188)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5180..5989
                     /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(5247..5266)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      6132..6719
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6621..6640
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6873..6890
                     /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 ggtccttcta gaaagctact gtcttctatc gaacaagcat gcgatatttg
      781 ccgacttaaa aagctcaagt gctccaaaga aaaaccgaag tgcgccaagt gtctgaagaa
      841 caactgggag tgtcgctact ctcccaaaac caaaaggtct ccgctgacta gggcacatct
      901 gacagaagtg gaatcaaggc tagaaagact ggaacagcta tttctactga tttttcctcg
      961 agaagacctt gacatgattt tgaaaatgga ttctttacag gatataaaag cattgttaac
     1021 aggattattt gtacaagata atgtgaataa agatgccgtc acagatagat tggcttcagt
     1081 ggagactgat atgcctctaa cattgagaca gcatagaata agtgcgacat catcatcgga
     1141 agagagtagt aacaaaggtc aaagacagtt gactgtatcg tcgaggtcga ccccgggaat
     1201 acagatctct cgagccgccc ccccgaccga tgtcagcctg ggggacgagc tccacttaga
     1261 cggcgaggac gtggcgatgg cgcatgccga cgcgctagac gatttcgatc tggacatgtt
     1321 gggggacggg gattccccgg gtccgggatt taccccccac gactccgccc cctacggcgc
     1381 tctggatatg taaaactagt aattaagtct cagccaccgt taactgaaca tgtcaaaacc
     1441 tgtggagact gttgagattt gatgttctga aaagataaag cctataaata aaatgttgcc
     1501 caaatttcct gcctgatgtt tttctttgtc tttgctacat ggctttgctg ctcggatcgg
     1561 ctcactctgt gtatgccacg ttcactttgt actctccttc tcacggtagg tttattattt
     1621 ttagatgtgc agttagtttc tgtgaaataa cacaccacac actgatattg tctgtgcatt
     1681 gacttggtga gtgcacattg tttttgatct tgacatattt atatttgatt gatcaggtga
     1741 actgtgtgaa tctaaagtgc tccatacaga tgttctgcat tgaaaatatt ctcattttat
     1801 tagtggaagt gagtgtatgc cacatccaat caatttcagc aaacacccca gtatgattta
     1861 atgcaaaaaa atgaaggtat caaacacgca ttactacttt gcagttaaat atttaacatt
     1921 tattccaaca cgaaaaaaag cagtaaataa cactttgaca aacacgtcag gacatcttat
     1981 ttttgtcacc ctcacaggca atttagtata atatattata tatatatata tatcatataa
     2041 taatattcag tataatatat atatatatat catattataa tattcagtat aatataaaac
     2101 acaaacacat atatgtataa tataatataa catttttatt tattgagatg cctctatgga
     2161 ccgtgttata agaagtaaag atcaggagaa gtaaacatga agtgtaatta tgaatactga
     2221 tgttaaatta agctatgatg agttttcact gttaatttac catctcaatt aaatgttgat
     2281 gcctccatga ccaagttaag cagatgagac tgaggacaac tgtagaagac aagatgttca
     2341 ctttgctgaa tatagctggc ttgacagtta tctatgactc tataaatata tatatatttt
     2401 ttttttataa aatgatttat ttataactat atatccattt ctcagacagg tgcttcatat
     2461 ccctcactcc cgtagctgtc catgctggat ctgtccccgt tgtttttaaa aagctaaata
     2521 agttattaac atgactgcat ccagcgagcc aaacctgtct ggtgtacagc taccagagaa
     2581 gcttgagatc ctagtcaccg gcgttcctag ttctttaaac tgttacaaag tgtttggacc
     2641 agcaactgaa ttgcactgta aattaccaca tcagggtctc cggggtcaac gctgctaagc
     2701 aatggactgg aaaatttgtg acattgagtt tttggaataa ctgggaatgc tggataactc
     2761 tttgggtaaa gctatggaac aaaagtacca gcatgaacta agaaatgact gtcacagcac
     2821 aacatcaaac acaatgcaag acaaaggttc tctgcttttg ccttacttga cagaaaaagt
     2881 aagctctagg ttcatcgcat gcagacagca gcaatacatg acatatataa aggctggctc
     2941 ttttctccct tccattgaac tgaaacttcc actcagtcag acttgcgggg gatccacagc
     3001 ctgactaact cgacaaaccc ccccaaacct aaggtgagtt gatctttaag ctttttacat
     3061 tttcagctcg catatatcaa ttcgcgcgca ttgtcacact agcacccact agcggtcaga
     3121 ctgcagattg cagcacgaaa caggaagctg actccacatg gtcacatgct cactgaagtg
     3181 ttgacttccc tgacagctgt gcactttcta aaccggtttt ctcattcatt tacagttcag
     3241 ccggaattca cctcgagacc atggctccaa agaagaagcg taaggtaacc atggcctcct
     3301 ccgaggacgt catcaaggag ttcatgcgct tcaaggtgcg catggagggc tccgtgaacg
     3361 gccacgagtt cgagatcgag ggcgagggcg agggccgccc ctacgagggc acccagaccg
     3421 ccaagctgaa ggtgaccaag ggcggccccc tgcccttcgc ctgggacatc ctgtcccctc
     3481 agttccagta cggctccaag gcctacgtga agcaccccgc cgacatcccc gactacttga
     3541 agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag gacggcggcg
     3601 tggtgaccgt gacccaggac tcctccctgc aggacggcga gttcatctac aaggtgaagc
     3661 tgcgcggcac caacttcccc tccgacggcc ccgtaatgca gaagaagacc atgggctggg
     3721 aggcctccac cgagcggatg taccccgagg acggcgccct gaagggcgag atcaagatga
     3781 ggctgaagct gaaggacggc ggccactacg acgccgaggt caagaccacc tacatggcca
     3841 agaagcccgt gcagctgccc ggcgcctaca agaccgacat caagctggac atcacctccc
     3901 acaacgagga ctacaccatc gtggaacagt acgagcgcgc cgagggccgc cactccaccg
     3961 gcgcctaaag ctgtgatctg tgccttctag ttgccagcca tctgttgttt gcccctcccc
     4021 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga
     4081 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
     4141 cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat
     4201 ggcttctgag gcggaaagaa ccaaagggcg aattaattca ttgatgagtt tggacaaacc
     4261 acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta
     4321 tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg
     4381 tttcaggttc agggggaggt gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt
     4441 ggtcattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg
     4501 tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata aacaagttaa
     4561 caacaacaat tgcattcatt ttatgtttca ggttcagggg gaggtgtggg aggtttttta
     4621 aagcaagtaa aacctctaca aatgtggtca ttcagatcaa ttaaccctca cccgggctag
     4681 cgatgcatgg gagctctgac gtacgttgtc tagcaaggaa gtgaagagct atccggatat
     4741 atatgtatgt tatcataact gtactggcta tgtcaggtgg taatgactgt aacgttacgt
     4801 tactcgttgt aggcacgaca ttgaatatgc tcttctccgc tgtggcccga acgcctcccg
     4861 ctagcgatgg gatctaatac gactcactat agggagacca agggaaaata gaatgaagtg
     4921 atctccaaaa aataagtact ttttgactgt aaataaaatt gtaaggagta aaaagtactt
     4981 ttttttctaa aaaaatgtaa ttaagtaaaa gtaaaagtat tgatttttaa ttgtactcaa
     5041 gtaaagtaaa aatccccaaa aataatactt aagtacagta atcaagtaaa attactcaag
     5101 tactttacac ctctgggatc cagctgcatc atgaacaata aaactgtctg cttacataaa
     5161 cagtaataca aggggtgtta tgagccatat tcaacgggaa acgtcgaggc cgcgattaaa
     5221 ttccaacatg gatgctgatt tatatgggta taaatgggct cgcgataatg tcgggcaatc
     5281 aggtgcgaca atctatcgct tgtatgggaa gcccgatgcg ccagagttgt ttctgaaaca
     5341 tggcaaaggt agcgttgcca atgatgttac agatgagatg gtcagactaa actggctgac
     5401 ggaatttatg cctcttccga ccatcaagca ttttatccgt actcctgatg atgcatggtt
     5461 actcaccact gcgatccccg gaaaaacagc attccaggta ttagaagaat atcctgattc
     5521 aggtgaaaat attgttgatg cgctggcagt gttcctgcgc cggttgcatt cgattcctgt
     5581 ttgtaattgt ccttttaaca gcgatcgcgt atttcgtctc gctcaggcgc aatcacgaat
     5641 gaataacggt ttggttgatg cgagtgattt tgatgacgag cgtaatggct ggcctgttga
     5701 acaagtctgg aaagaaatgc ataaactttt gccattctca ccggattcag tcgtcactca
     5761 tggtgatttc tcacttgata accttatttt tgacgagggg aaattaatag gttgtattga
     5821 tgttggacga gtcggaatcg cagaccgata ccaggatctt gccatcctat ggaactgcct
     5881 cggtgagttt tctccttcat tacagaaacg gctttttcaa aaatatggta ttgataatcc
     5941 tgatatgaat aaattgcagt ttcatttgat gctcgatgag tttttctaat cagaattggt
     6001 taattggttg taacactggc aaaaaggatc taggtgaaga tcctttttga taatctcatg
     6061 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     6121 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     6181 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     6241 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta
     6301 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     6361 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     6421 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     6481 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     6541 gttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     6601 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt gggtttcgcc
     6661 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     6721 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     6781 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     6841 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaac
     6901 agattt
//
