LOCUS       sequence-227663-        6820 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 6820)
  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 6820)
  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-cmlc2-TagBFP
FEATURES             Location/Qualifiers
     source          1..6820
                     /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             3413..3433
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3437..4138
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     polyA_signal    4148..4372
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    4442..4523
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    4647..4728
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        5045..5063
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(5086..5273,5274..5285)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(5339..5358)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5350..6159
                     /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(5417..5436)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      join(6302..6820,1..70)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6791..6810
                     /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 gcattcatcc atccttttca
     2881 tccctcaaat ctctcattca cgtccccctc cccatctgca cactttatct cattttccac
     2941 cctgctggaa tctgagcact tgtgcagtta tcagggctcc tgtatttagg aggctctggg
     3001 tgtccatgta ggggacgaac agaaacactg cagaccttta tagaagaaca aatgataaga
     3061 gtcctcatac ataaagactc cattagaaac gtcagtgacc caggagccca gaccaacagc
     3121 aaagcagaca gtgaaggatc cacagcctga ctaactcgac aaaccccccc aaacctaagg
     3181 tgagttgatc tttaagcttt ttacattttc agctcgcata tatcaattcg cgcgcattgt
     3241 cacactagca cccactagcg gtcagactgc agattgcagc acgaaacagg aagctgactc
     3301 cacatggtca catgctcact gaagtgttga cttccctgac agctgtgcac tttctaaacc
     3361 ggttttctca ttcatttaca gttcagccgg aattcacctc gagaccatgg ctccaaagaa
     3421 gaagcgtaag gtaaccatga gcgagctgat taaggagaac atgcacatga agctgtacat
     3481 ggagggcacc gtggacaacc atcacttcaa gtgcacatcc gagggcgaag gcaagcccta
     3541 cgagggcacc cagaccatga gaatcaaggt ggtcgagggc ggccctctcc ccttcgcctt
     3601 cgacatcctg gctactagct tcctctacgg cagcaagacc ttcatcaacc acacccaggg
     3661 catccccgac ttcttcaagc agtccttccc tgagggcttc acatgggaga gagtcaccac
     3721 atacgaagac gggggcgtgc tgaccgctac ccaggacacc agcctccagg acggctgcct
     3781 catctacaac gtcaagatca gaggggtgaa cttcacatcc aacggccctg tgatgcagaa
     3841 gaaaacactc ggctgggagg ccttcaccga gacgctgtac cccgctgacg gcggcctgga
     3901 aggcagaaac gacatggccc tgaagctcgt gggcgggagc catctgatcg caaacatcaa
     3961 gaccacatat agatccaaga aacccgctaa gaacctcaag atgcctggcg tctactatgt
     4021 ggactacaga ctggaaagaa tcaaggaggc caacaacgag acctacgtcg agcagcacga
     4081 ggtggcagtg gccagatact gcgacctccc tagcaaactg gggcacaagc tcaattaaag
     4141 ctgagatctg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc
     4201 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg
     4261 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg
     4321 gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag
     4381 gcggaaagaa ccaaagggcg aattaattca ttgatgagtt tggacaaacc acaactagaa
     4441 tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta tttgtaacca
     4501 ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg tttcaggttc
     4561 agggggaggt gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt ggtcattgat
     4621 gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt
     4681 gatgctattg ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat
     4741 tgcattcatt ttatgtttca ggttcagggg gaggtgtggg aggtttttta aagcaagtaa
     4801 aacctctaca aatgtggtca ttcagatcaa ttaaccctca cccgggctag cgatgcatgg
     4861 gagctctgac gtacgttgtc tagcaaggaa gtgaagagct atccggatat atatgtatgt
     4921 tatcataact gtactggcta tgtcaggtgg taatgactgt aacgttacgt tactcgttgt
     4981 aggcacgaca ttgaatatgc tcttctccgc tgtggcccga acgcctcccg ctagcgatgg
     5041 gatctaatac gactcactat agggagacca agggaaaata gaatgaagtg atctccaaaa
     5101 aataagtact ttttgactgt aaataaaatt gtaaggagta aaaagtactt ttttttctaa
     5161 aaaaatgtaa ttaagtaaaa gtaaaagtat tgatttttaa ttgtactcaa gtaaagtaaa
     5221 aatccccaaa aataatactt aagtacagta atcaagtaaa attactcaag tactttacac
     5281 ctctgggatc cagctgcatc atgaacaata aaactgtctg cttacataaa cagtaataca
     5341 aggggtgtta tgagccatat tcaacgggaa acgtcgaggc cgcgattaaa ttccaacatg
     5401 gatgctgatt tatatgggta taaatgggct cgcgataatg tcgggcaatc aggtgcgaca
     5461 atctatcgct tgtatgggaa gcccgatgcg ccagagttgt ttctgaaaca tggcaaaggt
     5521 agcgttgcca atgatgttac agatgagatg gtcagactaa actggctgac ggaatttatg
     5581 cctcttccga ccatcaagca ttttatccgt actcctgatg atgcatggtt actcaccact
     5641 gcgatccccg gaaaaacagc attccaggta ttagaagaat atcctgattc aggtgaaaat
     5701 attgttgatg cgctggcagt gttcctgcgc cggttgcatt cgattcctgt ttgtaattgt
     5761 ccttttaaca gcgatcgcgt atttcgtctc gctcaggcgc aatcacgaat gaataacggt
     5821 ttggttgatg cgagtgattt tgatgacgag cgtaatggct ggcctgttga acaagtctgg
     5881 aaagaaatgc ataaactttt gccattctca ccggattcag tcgtcactca tggtgatttc
     5941 tcacttgata accttatttt tgacgagggg aaattaatag gttgtattga tgttggacga
     6001 gtcggaatcg cagaccgata ccaggatctt gccatcctat ggaactgcct cggtgagttt
     6061 tctccttcat tacagaaacg gctttttcaa aaatatggta ttgataatcc tgatatgaat
     6121 aaattgcagt ttcatttgat gctcgatgag tttttctaat cagaattggt taattggttg
     6181 taacactggc aaaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     6241 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     6301 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     6361 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     6421 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     6481 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     6541 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     6601 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     6661 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg gttcccgaag
     6721 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     6781 agcttccagg gggaaacgcc tggtatcttt atagtcctgt
//
