LOCUS       sequence-229893-        6243 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 6243)
  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 6243)
  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-Stop-gcry-TagBFP
FEATURES             Location/Qualifiers
     source          1..6243
                     /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..699
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWTCHTSIMIGSND
                     "
     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             2579..2599
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2603..3304
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     polyA_signal    3314..3538
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    3608..3689
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    3813..3894
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        4211..4229
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(4252..4439,4440..4451)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(4505..4524)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4516..5325
                     /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(4583..4602)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      5468..6056
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5957..5976
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6210..6227
                     /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 cttctataat gataggtagt aatgattagt aatgagacta gtaattaagt
      721 ctcagccacc gttaactgaa catgtcaaaa cctgtggaga ctgttgagat ttgatgttct
      781 gaaaagataa agcctataaa taaaatgttg cccaaatttc ctgcctgatg tttttctttg
      841 tctttgctac atggctttgc tgctcggatc ggctcactct gtgtatgcca cgttcacttt
      901 gtactctcct tctcacggta ggtttattat ttttagatgt gcagttagtt tctgtgaaat
      961 aacacaccac acactgatat tgtctgtgca ttgacttggt gagtgcacat tgtttttgat
     1021 cttgacatat ttatatttga ttgatcaggt gaactgtgtg aatctaaagt gctccataca
     1081 gatgttctgc attgaaaata ttctcatttt attagtggaa gtgagtgtat gccacatcca
     1141 atcaatttca gcaaacaccc cagtatgatt taatgcaaaa aaatgaaggt atcaaacacg
     1201 cattactact ttgcagttaa atatttaaca tttattccaa cacgaaaaaa agcagtaaat
     1261 aacactttga caaacacgtc aggacatctt atttttgtca ccctcacagg caatttagta
     1321 taatatatta tatatatata tatatcatat aataatattc agtataatat atatatatat
     1381 atcatattat aatattcagt ataatataaa acacaaacac atatatgtat aatataatat
     1441 aacattttta tttattgaga tgcctctatg gaccgtgtta taagaagtaa agatcaggag
     1501 aagtaaacat gaagtgtaat tatgaatact gatgttaaat taagctatga tgagttttca
     1561 ctgttaattt accatctcaa ttaaatgttg atgcctccat gaccaagtta agcagatgag
     1621 actgaggaca actgtagaag acaagatgtt cactttgctg aatatagctg gcttgacagt
     1681 tatctatgac tctataaata tatatatatt ttttttttat aaaatgattt atttataact
     1741 atatatccat ttctcagaca ggtgcttcat atccctcact cccgtagctg tccatgctgg
     1801 atctgtcccc gttgttttta aaaagctaaa taagttatta acatgactgc atccagcgag
     1861 ccaaacctgt ctggtgtaca gctaccagag aagcttgaga tcctagtcac cggcgttcct
     1921 agttctttaa actgttacaa agtgtttgga ccagcaactg aattgcactg taaattacca
     1981 catcagggtc tccggggtca acgctgctaa gcaatggact ggaaaatttg tgacattgag
     2041 tttttggaat aactgggaat gctggataac tctttgggta aagctatgga acaaaagtac
     2101 cagcatgaac taagaaatga ctgtcacagc acaacatcaa acacaatgca agacaaaggt
     2161 tctctgcttt tgccttactt gacagaaaaa gtaagctcta ggttcatcgc atgcagacag
     2221 cagcaataca tgacatatat aaaggctggc tcttttctcc cttccattga actgaaactt
     2281 ccactcagtc agacttgcgg gggatccaca gcctgactaa ctcgacaaac ccccccaaac
     2341 ctaaggtgag ttgatcttta agctttttac attttcagct cgcatatatc aattcgcgcg
     2401 cattgtcaca ctagcaccca ctagcggtca gactgcagat tgcagcacga aacaggaagc
     2461 tgactccaca tggtcacatg ctcactgaag tgttgacttc cctgacagct gtgcactttc
     2521 taaaccggtt ttctcattca tttacagttc agccggaatt cacctcgaga ccatggctcc
     2581 aaagaagaag cgtaaggtaa ccatgagcga gctgattaag gagaacatgc acatgaagct
     2641 gtacatggag ggcaccgtgg acaaccatca cttcaagtgc acatccgagg gcgaaggcaa
     2701 gccctacgag ggcacccaga ccatgagaat caaggtggtc gagggcggcc ctctcccctt
     2761 cgccttcgac atcctggcta ctagcttcct ctacggcagc aagaccttca tcaaccacac
     2821 ccagggcatc cccgacttct tcaagcagtc cttccctgag ggcttcacat gggagagagt
     2881 caccacatac gaagacgggg gcgtgctgac cgctacccag gacaccagcc tccaggacgg
     2941 ctgcctcatc tacaacgtca agatcagagg ggtgaacttc acatccaacg gccctgtgat
     3001 gcagaagaaa acactcggct gggaggcctt caccgagacg ctgtaccccg ctgacggcgg
     3061 cctggaaggc agaaacgaca tggccctgaa gctcgtgggc gggagccatc tgatcgcaaa
     3121 catcaagacc acatatagat ccaagaaacc cgctaagaac ctcaagatgc ctggcgtcta
     3181 ctatgtggac tacagactgg aaagaatcaa ggaggccaac aacgagacct acgtcgagca
     3241 gcacgaggtg gcagtggcca gatactgcga cctccctagc aaactggggc acaagctcaa
     3301 ttaaagctga gatctgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
     3361 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
     3421 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
     3481 aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct
     3541 tctgaggcgg aaagaaccaa agggcgaatt aattcattga tgagtttgga caaaccacaa
     3601 ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg
     3661 taaccattat aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc
     3721 aggttcaggg ggaggtgtgg gaggtttttt aaagcaagta aaacctctac aaatgtggtc
     3781 attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa
     3841 atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca agttaacaac
     3901 aacaattgca ttcattttat gtttcaggtt cagggggagg tgtgggaggt tttttaaagc
     3961 aagtaaaacc tctacaaatg tggtcattca gatcaattaa ccctcacccg ggctagcgat
     4021 gcatgggagc tctgacgtac gttgtctagc aaggaagtga agagctatcc ggatatatat
     4081 gtatgttatc ataactgtac tggctatgtc aggtggtaat gactgtaacg ttacgttact
     4141 cgttgtaggc acgacattga atatgctctt ctccgctgtg gcccgaacgc ctcccgctag
     4201 cgatgggatc taatacgact cactataggg agaccaaggg aaaatagaat gaagtgatct
     4261 ccaaaaaata agtacttttt gactgtaaat aaaattgtaa ggagtaaaaa gtactttttt
     4321 ttctaaaaaa atgtaattaa gtaaaagtaa aagtattgat ttttaattgt actcaagtaa
     4381 agtaaaaatc cccaaaaata atacttaagt acagtaatca agtaaaatta ctcaagtact
     4441 ttacacctct gggatccagc tgcatcatga acaataaaac tgtctgctta cataaacagt
     4501 aatacaaggg gtgttatgag ccatattcaa cgggaaacgt cgaggccgcg attaaattcc
     4561 aacatggatg ctgatttata tgggtataaa tgggctcgcg ataatgtcgg gcaatcaggt
     4621 gcgacaatct atcgcttgta tgggaagccc gatgcgccag agttgtttct gaaacatggc
     4681 aaaggtagcg ttgccaatga tgttacagat gagatggtca gactaaactg gctgacggaa
     4741 tttatgcctc ttccgaccat caagcatttt atccgtactc ctgatgatgc atggttactc
     4801 accactgcga tccccggaaa aacagcattc caggtattag aagaatatcc tgattcaggt
     4861 gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt tgcattcgat tcctgtttgt
     4921 aattgtcctt ttaacagcga tcgcgtattt cgtctcgctc aggcgcaatc acgaatgaat
     4981 aacggtttgg ttgatgcgag tgattttgat gacgagcgta atggctggcc tgttgaacaa
     5041 gtctggaaag aaatgcataa acttttgcca ttctcaccgg attcagtcgt cactcatggt
     5101 gatttctcac ttgataacct tatttttgac gaggggaaat taataggttg tattgatgtt
     5161 ggacgagtcg gaatcgcaga ccgataccag gatcttgcca tcctatggaa ctgcctcggt
     5221 gagttttctc cttcattaca gaaacggctt tttcaaaaat atggtattga taatcctgat
     5281 atgaataaat tgcagtttca tttgatgctc gatgagtttt tctaatcaga attggttaat
     5341 tggttgtaac actggcaaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     5401 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     5461 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     5521 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     5581 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc
     5641 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     5701 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     5761 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     5821 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacggttc
     5881 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     5941 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     6001 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     6061 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     6121 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
     6181 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaacaga
     6241 ttt
//
