LOCUS       sequence-229912-        6913 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 6913)
  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 6913)
  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-Fuse-gcry-TagBFP
FEATURES             Location/Qualifiers
     source          1..6913
                     /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..687
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWTCHTSRRR"
     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             701..1375
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="ASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAKL
                     KVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVV
                     TVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKMR
                     LKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHST
                     GA"
     CDS             701..1375
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /note="mammalian codon-optimized"
                     /translation="ASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAKL
                     KVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVV
                     TVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKMR
                     LKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHST
                     GA"
     primer_bind     complement(828..846)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1096..1115
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1290..1313
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     CDS             3249..3269
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3273..3974
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     polyA_signal    3984..4208
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    4278..4359
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    4483..4564
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        4881..4899
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(4922..5109,5110..5121)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(5175..5194)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5186..5995
                     /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(5253..5272)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      6138..6726
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6627..6646
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6880..6897
                     /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 cttctaggag gaggtagcgg aggtagcgga gcctcctccg aggacgtcat
      721 caaggagttc atgcgcttca aggtgcgcat ggagggctcc gtgaacggcc acgagttcga
      781 gatcgagggc gagggcgagg gccgccccta cgagggcacc cagaccgcca agctgaaggt
      841 gaccaagggc ggccccctgc ccttcgcctg ggacatcctg tcccctcagt tccagtacgg
      901 ctccaaggcc tacgtgaagc accccgccga catccccgac tacttgaagc tgtccttccc
      961 cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac
     1021 ccaggactcc tccctgcagg acggcgagtt catctacaag gtgaagctgc gcggcaccaa
     1081 cttcccctcc gacggccccg taatgcagaa gaagaccatg ggctgggagg cctccaccga
     1141 gcggatgtac cccgaggacg gcgccctgaa gggcgagatc aagatgaggc tgaagctgaa
     1201 ggacggcggc cactacgacg ccgaggtcaa gaccacctac atggccaaga agcccgtgca
     1261 gctgcccggc gcctacaaga ccgacatcaa gctggacatc acctcccaca acgaggacta
     1321 caccatcgtg gaacagtacg agcgcgccga gggccgccac tccaccggag cctaagacta
     1381 gtaattaagt ctcagccacc gttaactgaa catgtcaaaa cctgtggaga ctgttgagat
     1441 ttgatgttct gaaaagataa agcctataaa taaaatgttg cccaaatttc ctgcctgatg
     1501 tttttctttg tctttgctac atggctttgc tgctcggatc ggctcactct gtgtatgcca
     1561 cgttcacttt gtactctcct tctcacggta ggtttattat ttttagatgt gcagttagtt
     1621 tctgtgaaat aacacaccac acactgatat tgtctgtgca ttgacttggt gagtgcacat
     1681 tgtttttgat cttgacatat ttatatttga ttgatcaggt gaactgtgtg aatctaaagt
     1741 gctccataca gatgttctgc attgaaaata ttctcatttt attagtggaa gtgagtgtat
     1801 gccacatcca atcaatttca gcaaacaccc cagtatgatt taatgcaaaa aaatgaaggt
     1861 atcaaacacg cattactact ttgcagttaa atatttaaca tttattccaa cacgaaaaaa
     1921 agcagtaaat aacactttga caaacacgtc aggacatctt atttttgtca ccctcacagg
     1981 caatttagta taatatatta tatatatata tatatcatat aataatattc agtataatat
     2041 atatatatat atcatattat aatattcagt ataatataaa acacaaacac atatatgtat
     2101 aatataatat aacattttta tttattgaga tgcctctatg gaccgtgtta taagaagtaa
     2161 agatcaggag aagtaaacat gaagtgtaat tatgaatact gatgttaaat taagctatga
     2221 tgagttttca ctgttaattt accatctcaa ttaaatgttg atgcctccat gaccaagtta
     2281 agcagatgag actgaggaca actgtagaag acaagatgtt cactttgctg aatatagctg
     2341 gcttgacagt tatctatgac tctataaata tatatatatt ttttttttat aaaatgattt
     2401 atttataact atatatccat ttctcagaca ggtgcttcat atccctcact cccgtagctg
     2461 tccatgctgg atctgtcccc gttgttttta aaaagctaaa taagttatta acatgactgc
     2521 atccagcgag ccaaacctgt ctggtgtaca gctaccagag aagcttgaga tcctagtcac
     2581 cggcgttcct agttctttaa actgttacaa agtgtttgga ccagcaactg aattgcactg
     2641 taaattacca catcagggtc tccggggtca acgctgctaa gcaatggact ggaaaatttg
     2701 tgacattgag tttttggaat aactgggaat gctggataac tctttgggta aagctatgga
     2761 acaaaagtac cagcatgaac taagaaatga ctgtcacagc acaacatcaa acacaatgca
     2821 agacaaaggt tctctgcttt tgccttactt gacagaaaaa gtaagctcta ggttcatcgc
     2881 atgcagacag cagcaataca tgacatatat aaaggctggc tcttttctcc cttccattga
     2941 actgaaactt ccactcagtc agacttgcgg gggatccaca gcctgactaa ctcgacaaac
     3001 ccccccaaac ctaaggtgag ttgatcttta agctttttac attttcagct cgcatatatc
     3061 aattcgcgcg cattgtcaca ctagcaccca ctagcggtca gactgcagat tgcagcacga
     3121 aacaggaagc tgactccaca tggtcacatg ctcactgaag tgttgacttc cctgacagct
     3181 gtgcactttc taaaccggtt ttctcattca tttacagttc agccggaatt cacctcgaga
     3241 ccatggctcc aaagaagaag cgtaaggtaa ccatgagcga gctgattaag gagaacatgc
     3301 acatgaagct gtacatggag ggcaccgtgg acaaccatca cttcaagtgc acatccgagg
     3361 gcgaaggcaa gccctacgag ggcacccaga ccatgagaat caaggtggtc gagggcggcc
     3421 ctctcccctt cgccttcgac atcctggcta ctagcttcct ctacggcagc aagaccttca
     3481 tcaaccacac ccagggcatc cccgacttct tcaagcagtc cttccctgag ggcttcacat
     3541 gggagagagt caccacatac gaagacgggg gcgtgctgac cgctacccag gacaccagcc
     3601 tccaggacgg ctgcctcatc tacaacgtca agatcagagg ggtgaacttc acatccaacg
     3661 gccctgtgat gcagaagaaa acactcggct gggaggcctt caccgagacg ctgtaccccg
     3721 ctgacggcgg cctggaaggc agaaacgaca tggccctgaa gctcgtgggc gggagccatc
     3781 tgatcgcaaa catcaagacc acatatagat ccaagaaacc cgctaagaac ctcaagatgc
     3841 ctggcgtcta ctatgtggac tacagactgg aaagaatcaa ggaggccaac aacgagacct
     3901 acgtcgagca gcacgaggtg gcagtggcca gatactgcga cctccctagc aaactggggc
     3961 acaagctcaa ttaaagctga gatctgtgcc ttctagttgc cagccatctg ttgtttgccc
     4021 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa
     4081 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg
     4141 gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg
     4201 ctctatggct tctgaggcgg aaagaaccaa agggcgaatt aattcattga tgagtttgga
     4261 caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt
     4321 gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa ttgcattcat
     4381 tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt aaagcaagta aaacctctac
     4441 aaatgtggtc attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt
     4501 tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca
     4561 agttaacaac aacaattgca ttcattttat gtttcaggtt cagggggagg tgtgggaggt
     4621 tttttaaagc aagtaaaacc tctacaaatg tggtcattca gatcaattaa ccctcacccg
     4681 ggctagcgat gcatgggagc tctgacgtac gttgtctagc aaggaagtga agagctatcc
     4741 ggatatatat gtatgttatc ataactgtac tggctatgtc aggtggtaat gactgtaacg
     4801 ttacgttact cgttgtaggc acgacattga atatgctctt ctccgctgtg gcccgaacgc
     4861 ctcccgctag cgatgggatc taatacgact cactataggg agaccaaggg aaaatagaat
     4921 gaagtgatct ccaaaaaata agtacttttt gactgtaaat aaaattgtaa ggagtaaaaa
     4981 gtactttttt ttctaaaaaa atgtaattaa gtaaaagtaa aagtattgat ttttaattgt
     5041 actcaagtaa agtaaaaatc cccaaaaata atacttaagt acagtaatca agtaaaatta
     5101 ctcaagtact ttacacctct gggatccagc tgcatcatga acaataaaac tgtctgctta
     5161 cataaacagt aatacaaggg gtgttatgag ccatattcaa cgggaaacgt cgaggccgcg
     5221 attaaattcc aacatggatg ctgatttata tgggtataaa tgggctcgcg ataatgtcgg
     5281 gcaatcaggt gcgacaatct atcgcttgta tgggaagccc gatgcgccag agttgtttct
     5341 gaaacatggc aaaggtagcg ttgccaatga tgttacagat gagatggtca gactaaactg
     5401 gctgacggaa tttatgcctc ttccgaccat caagcatttt atccgtactc ctgatgatgc
     5461 atggttactc accactgcga tccccggaaa aacagcattc caggtattag aagaatatcc
     5521 tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt tgcattcgat
     5581 tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt cgtctcgctc aggcgcaatc
     5641 acgaatgaat aacggtttgg ttgatgcgag tgattttgat gacgagcgta atggctggcc
     5701 tgttgaacaa gtctggaaag aaatgcataa acttttgcca ttctcaccgg attcagtcgt
     5761 cactcatggt gatttctcac ttgataacct tatttttgac gaggggaaat taataggttg
     5821 tattgatgtt ggacgagtcg gaatcgcaga ccgataccag gatcttgcca tcctatggaa
     5881 ctgcctcggt gagttttctc cttcattaca gaaacggctt tttcaaaaat atggtattga
     5941 taatcctgat atgaataaat tgcagtttca tttgatgctc gatgagtttt tctaatcaga
     6001 attggttaat tggttgtaac actggcaaaa aggatctagg tgaagatcct ttttgataat
     6061 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     6121 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     6181 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     6241 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
     6301 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     6361 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     6421 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     6481 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     6541 gccacggttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     6601 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     6661 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     6721 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     6781 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     6841 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     6901 gcggaacaga ttt
//
