LOCUS       sequence-353839-        7285 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor for generating conditional alleles in zebrafish using precise 
            CRISPR directed genomic integration with the GeneWeld method.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7285)
  AUTHORS   Liu F, Kambakam S, Almeida MP, Ming Z, Welker JM, Wierson WA, 
            Schultz-Rogers LE, Ekker SC, Clark KJ, Essner JJ, McGrail M
  TITLE     Cre/lox regulated conditional rescue and inactivation with zebrafish
            UFlip alleles generated by CRISPR-Cas9 targeted integration.
  JOURNAL   Elife. 2022 Jun 17;11. pii: 71478. doi: 10.7554/eLife.71478.
  PUBMED    35713402
REFERENCE   2  (bases 1 to 7285)
  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: "2022-06-17"; volume: "11. pii: 71478. doi"; pages: " 
            10.7554/eLife.71478"
COMMENT     Sequence Label: pUFlip-floxed2A-KalTA4-; HE1A:BFP -1
FEATURES             Location/Qualifiers
     source          1..7285
                     /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..953
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWHLPHFRPHNFK"
     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"
     protein_bind    941..972
                     /label=rox
                     /bound_moiety="Dre recombinase"
     protein_bind    complement(978..1011)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    complement(1016..1049)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     CDS             1196..1252
                     /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             1253..1693
                     /codon_start=1
                     /gene="Saccharomyces cerevisiae GAL4 (truncated)"
                     /product="DNA binding domain of the GAL4 transcriptional 
                     activator"
                     /label=GAL4 DNA binding domain
                     /translation="MKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTK
                     RSPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNK
                     DAVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVS"
     CDS             3434..3454
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3458..4159
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     protein_bind    5234..5267
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    5273..5306
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    5313..5344
                     /label=rox
                     /bound_moiety="Dre recombinase"
     promoter        5510..5528
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(5551..5738,5739..5750)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(5804..5823)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5815..6624
                     /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(5882..5901)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      join(6767..7285,1..70)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7256..7275
                     /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 cgaatggcac ctgccacact tccggccgca taactttaaa taatgccaat
      961 tatttaaagt tactatcata acttcgtata gcatacatta tacgaagtta tcaccataac
     1021 ttcgtatagg atactttata cgaagttatc ctaggcttac cacccactag cggtcagact
     1081 gcagattgca gcacgaaaca ggaagctgac tccacatggt cacatgctca ctgaagtgtt
     1141 gacttccctg acagctgtgc actttctaaa ccggttttct cattcattta cagttgccac
     1201 gaacttctct ctgttaaagc aagcaggaga cgtggaagaa aaccccggtc ctatgaaact
     1261 gctctcatcc atcgagcaag cctgcgacat ttgtcggctt aagaagctga aatgctccaa
     1321 ggaaaagccg aaatgtgcca aatgcctgaa gaacaattgg gaatgtcgtt actctcccaa
     1381 aaccaagcga agtccactca caagggctca tctgaccgaa gtggagagca ggctagagag
     1441 actggaacaa ctctttttgc tcatcttccc tagagaggac cttgacatga tcctcaagat
     1501 ggattctctc caggatatta aagccctttt gactggctta ttcgtccagg acaatgtgaa
     1561 caaagacgct gtgacagacc gattggcaag tgtcgagacc gatatgcctc tgacactgag
     1621 acagcacaga atcagcgcta cttcctcaag cgaagagtct tctaacaagg gacagagaca
     1681 gctgactgtt tcgagcaggt cgaccccgtc cccggccgac gccctggacg acggcgacct
     1741 ggacatgctg cctgctgatg ctctcgatga tttcgatctg gatatgctcc cggccgacgc
     1801 cctggacgac tacgacctgg acatcctccc gggtaactaa taattaagtc tcagccaccg
     1861 ttaactgaac atgtcaaaac ctgtggagac tgttgagatt tgatgttctg aaaagataaa
     1921 gcctataaat aaaatgttgc ccaaatttcc tgcctgatgt ttttctttgt ctttgctaca
     1981 tggctttgct gctcggatcg gctcactctg tgtatgccac gttcactttg tactctcctt
     2041 ctcacggtag gtttattatt tttagatgtg cagttagttt ctgtgaaata acacaccaca
     2101 cactgatatt gtctgtgcat tgacttggtg agtgcacatt gtttttgatc ttgacatatt
     2161 tatatttgat tgatcaggtg aactgtgtga atctaaagtg ctccatacag atgttctgca
     2221 ttgaaaatat tctcatttta ttagtggaag tgagtgtatg ccacatccaa tcaatttcag
     2281 caaacacccc agtatgattt aatgcaaaaa aatgaaggta tcaaacacgc attactactt
     2341 tgcagttaaa tatttaacat ttattccaac acgaaaaaaa gcagtaaata acactttgac
     2401 aaacacgtca ggacatctta tttttgtcac cctcacaggc aatttagtat aatatattat
     2461 atatatatat atatcatata ataatattca gtataatata tatatatata tcatattata
     2521 atattcagta taatataaaa cacaaacaca tatatgtata atataatata acatttttat
     2581 ttattgagat gcctctatgg accgtgttat aagaagtaaa gatcaggaga agtaaacatg
     2641 aagtgtaatt atgaatactg atgttaaatt aagctatgat gagttttcac tgttaattta
     2701 ccatctcaat taaatgttga tgcctccatg accaagttaa gcagatgaga ctgaggacaa
     2761 ctgtagaaga caagatgttc actttgctga atatagctgg cttgacagtt atctatgact
     2821 ctataaatat atatatattt ttttttttat aaaatgattt atttataact atatatccat
     2881 ttctcagaca ggtgcttcat atccctcact cccgtagctg tccatgctgg atctgtcccc
     2941 gttgttttta aaaagctaaa taagttatta acatgactgc atccagcgag ccaaacctgt
     3001 ctggtgtaca gctaccagag aagcttgaga tcctagtcaa agctttcaac cactccaggc
     3061 atagctagag ccactgatgc atctatagag ctgaagtaaa gatagggttg atgcaaatcg
     3121 atttagaatt ttcaatgtag aaaaaacaaa ataaaaagtc tattcacttg atggatagat
     3181 tagtcaaaca aacatagagt aggcacttcc cttgactctg taaacttttg gatttaacag
     3241 attgtgcact ccttttagtt aaacacatta gtccctgaaa actaagaccc taaccataat
     3301 cccattgtag acccacctac tcgggtgaat ataaagctcg cacagatcat ctcacagaca
     3361 tcagaccttg gcaaaggagc atcctgaaca tctctacaca ctgaggcaac gaattcacct
     3421 cgagaccatg gctccaaaga agaagcgtaa ggtaaccatg agcgagctga ttaaggagaa
     3481 catgcacatg aagctgtaca tggagggcac cgtggacaac catcacttca agtgcacatc
     3541 cgagggcgaa ggcaagccct acgagggcac ccagaccatg agaatcaagg tggtcgaggg
     3601 cggccctctc cccttcgcct tcgacatcct ggctactagc ttcctctacg gcagcaagac
     3661 cttcatcaac cacacccagg gcatccccga cttcttcaag cagtccttcc ctgagggctt
     3721 cacatgggag agagtcacca catacgaaga cgggggcgtg ctgaccgcta cccaggacac
     3781 cagcctccag gacggctgcc tcatctacaa cgtcaagatc agaggggtga acttcacatc
     3841 caacggccct gtgatgcaga agaaaacact cggctgggag gccttcaccg agacgctgta
     3901 ccccgctgac ggcggcctgg aaggcagaaa cgacatggcc ctgaagctcg tgggcgggag
     3961 ccatctgatc gcaaacatca agaccacata tagatccaag aaacccgcta agaacctcaa
     4021 gatgcctggc gtctactatg tggactacag actggaaaga atcaaggagg ccaacaacga
     4081 gacctacgtc gagcagcacg aggtggcagt ggccagatac tgcgacctcc ctagcaaact
     4141 ggggcacaag ctcaattaaa gctgactaat ttatttagca gtagatagct atattgtgtg
     4201 aagtggtaac agtccgtaga tctacggact gttaccactt cacgccgact caaactgcgc
     4261 agagaaactt caaacgacaa cattggcatg gcttttgtta tttttggcgc ttgactcagg
     4321 atctaaaaac tggaacggtg aaggtgacgg caatgttttt tggcaaataa gcatccccga
     4381 agttctacaa tgcatctgag gactccatgt tttgttttct ttagtcattc caaatgtttg
     4441 ttaaatgcat tgttccgaaa cttatttgcc tctatgaagg ctgcccagta attgggagca
     4501 tacttaacat tgtagtattg tatgtaaatt atgtaacaaa acaatgtctg ggtttttgta
     4561 cttttcagcc ttaaaatctt gggtttattt ttttttcttt gttccaaaaa aacgaagctt
     4621 taccattcaa gatgtaaagg tttccattcc ccctgggcat attgtaaaag ctgtgtggaa
     4681 cgtggcggtg ccagacattt ggtggggcca acctgtacac tgactaattc aattccaata
     4741 aaagtgcaca tgtaagacat cctacccctg tctcatttcc tgtcttggct ctgagtggct
     4801 tcaataaaaa tggcttttgt gaagttgcac catgttaatt acatttcagc actttcacac
     4861 caggtttttg actaatttga atttatcgaa gtttgacctt tttttttttt tttcttcaag
     4921 ctaatttgag gttatcaaac catttcattg gaggaaactt aatgtttaaa agcataaatt
     4981 cgaaacatga ttcatttatt taactgctta atggtgaaac aaacttcaag taatacacca
     5041 agtgttccct catatgaagg tagctaactg caaacggcct taactttcca tcttaagttg
     5101 agctacataa aggctttaac aggaaggaac tgccagttta aaagtcaaac caccatgact
     5161 gatataaatt cttgtccata taaccttgcg tttcacacaa tatagctatc tactgctaaa
     5221 taaattagag ctcataactt cgtataatgt atgctatacg aagttatgtt agataacttc
     5281 gtataaagta tcctatacga agttatgcga gctaacttta aataattggc attatttaaa
     5341 gttatgaatc gataagtgaa gagctatccg gatatatatg tatgttatca taactgtact
     5401 ggctatgtca ggtggtaatg actgtaacgt tacgttactc gttgtaggca cgacattgaa
     5461 tatgctcttc tccgctgtgg cccgaacgcc tcccgctagc gatgggatct aatacgactc
     5521 actataggga gaccaaggga aaatagaatg aagtgatctc caaaaaataa gtactttttg
     5581 actgtaaata aaattgtaag gagtaaaaag tacttttttt tctaaaaaaa tgtaattaag
     5641 taaaagtaaa agtattgatt tttaattgta ctcaagtaaa gtaaaaatcc ccaaaaataa
     5701 tacttaagta cagtaatcaa gtaaaattac tcaagtactt tacacctctg ggatccagct
     5761 gcatcatgaa caataaaact gtctgcttac ataaacagta atacaagggg tgttatgagc
     5821 catattcaac gggaaacgtc gaggccgcga ttaaattcca acatggatgc tgatttatat
     5881 gggtataaat gggctcgcga taatgtcggg caatcaggtg cgacaatcta tcgcttgtat
     5941 gggaagcccg atgcgccaga gttgtttctg aaacatggca aaggtagcgt tgccaatgat
     6001 gttacagatg agatggtcag actaaactgg ctgacggaat ttatgcctct tccgaccatc
     6061 aagcatttta tccgtactcc tgatgatgca tggttactca ccactgcgat ccccggaaaa
     6121 acagcattcc aggtattaga agaatatcct gattcaggtg aaaatattgt tgatgcgctg
     6181 gcagtgttcc tgcgccggtt gcattcgatt cctgtttgta attgtccttt taacagcgat
     6241 cgcgtatttc gtctcgctca ggcgcaatca cgaatgaata acggtttggt tgatgcgagt
     6301 gattttgatg acgagcgtaa tggctggcct gttgaacaag tctggaaaga aatgcataaa
     6361 cttttgccat tctcaccgga ttcagtcgtc actcatggtg atttctcact tgataacctt
     6421 atttttgacg aggggaaatt aataggttgt attgatgttg gacgagtcgg aatcgcagac
     6481 cgataccagg atcttgccat cctatggaac tgcctcggtg agttttctcc ttcattacag
     6541 aaacggcttt ttcaaaaata tggtattgat aatcctgata tgaataaatt gcagtttcat
     6601 ttgatgctcg atgagttttt ctaatcagaa ttggttaatt ggttgtaaca ctggcaaaaa
     6661 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     6721 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     6781 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     6841 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     6901 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     6961 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     7021 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     7081 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     7141 gatacctaca gcgtgagcta tgagaaagcg ccacggttcc cgaagggaga aaggcggaca
     7201 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     7261 acgcctggta tctttatagt cctgt
//
