LOCUS       sequence-353840-        7283 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 7283)
  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 7283)
  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 -0
FEATURES             Location/Qualifiers
     source          1..7283
                     /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             1194..1250
                     /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             1251..1691
                     /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             3432..3452
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3456..4157
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     protein_bind    5232..5265
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    5271..5304
                     /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    5311..5342
                     /label=rox
                     /bound_moiety="Dre recombinase"
     promoter        5508..5526
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(5549..5736,5737..5748)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(5802..5821)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5813..6622
                     /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(5880..5899)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      join(6765..7283,1..70)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7254..7273
                     /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 caggccacga
     1201 acttctctct gttaaagcaa gcaggagacg tggaagaaaa ccccggtcct atgaaactgc
     1261 tctcatccat cgagcaagcc tgcgacattt gtcggcttaa gaagctgaaa tgctccaagg
     1321 aaaagccgaa atgtgccaaa tgcctgaaga acaattggga atgtcgttac tctcccaaaa
     1381 ccaagcgaag tccactcaca agggctcatc tgaccgaagt ggagagcagg ctagagagac
     1441 tggaacaact ctttttgctc atcttcccta gagaggacct tgacatgatc ctcaagatgg
     1501 attctctcca ggatattaaa gcccttttga ctggcttatt cgtccaggac aatgtgaaca
     1561 aagacgctgt gacagaccga ttggcaagtg tcgagaccga tatgcctctg acactgagac
     1621 agcacagaat cagcgctact tcctcaagcg aagagtcttc taacaaggga cagagacagc
     1681 tgactgtttc gagcaggtcg accccgtccc cggccgacgc cctggacgac ggcgacctgg
     1741 acatgctgcc tgctgatgct ctcgatgatt tcgatctgga tatgctcccg gccgacgccc
     1801 tggacgacta cgacctggac atcctcccgg gtaactaata attaagtctc agccaccgtt
     1861 aactgaacat gtcaaaacct gtggagactg ttgagatttg atgttctgaa aagataaagc
     1921 ctataaataa aatgttgccc aaatttcctg cctgatgttt ttctttgtct ttgctacatg
     1981 gctttgctgc tcggatcggc tcactctgtg tatgccacgt tcactttgta ctctccttct
     2041 cacggtaggt ttattatttt tagatgtgca gttagtttct gtgaaataac acaccacaca
     2101 ctgatattgt ctgtgcattg acttggtgag tgcacattgt ttttgatctt gacatattta
     2161 tatttgattg atcaggtgaa ctgtgtgaat ctaaagtgct ccatacagat gttctgcatt
     2221 gaaaatattc tcattttatt agtggaagtg agtgtatgcc acatccaatc aatttcagca
     2281 aacaccccag tatgatttaa tgcaaaaaaa tgaaggtatc aaacacgcat tactactttg
     2341 cagttaaata tttaacattt attccaacac gaaaaaaagc agtaaataac actttgacaa
     2401 acacgtcagg acatcttatt tttgtcaccc tcacaggcaa tttagtataa tatattatat
     2461 atatatatat atcatataat aatattcagt ataatatata tatatatatc atattataat
     2521 attcagtata atataaaaca caaacacata tatgtataat ataatataac atttttattt
     2581 attgagatgc ctctatggac cgtgttataa gaagtaaaga tcaggagaag taaacatgaa
     2641 gtgtaattat gaatactgat gttaaattaa gctatgatga gttttcactg ttaatttacc
     2701 atctcaatta aatgttgatg cctccatgac caagttaagc agatgagact gaggacaact
     2761 gtagaagaca agatgttcac tttgctgaat atagctggct tgacagttat ctatgactct
     2821 ataaatatat atatattttt ttttttataa aatgatttat ttataactat atatccattt
     2881 ctcagacagg tgcttcatat ccctcactcc cgtagctgtc catgctggat ctgtccccgt
     2941 tgtttttaaa aagctaaata agttattaac atgactgcat ccagcgagcc aaacctgtct
     3001 ggtgtacagc taccagagaa gcttgagatc ctagtcaaag ctttcaacca ctccaggcat
     3061 agctagagcc actgatgcat ctatagagct gaagtaaaga tagggttgat gcaaatcgat
     3121 ttagaatttt caatgtagaa aaaacaaaat aaaaagtcta ttcacttgat ggatagatta
     3181 gtcaaacaaa catagagtag gcacttccct tgactctgta aacttttgga tttaacagat
     3241 tgtgcactcc ttttagttaa acacattagt ccctgaaaac taagacccta accataatcc
     3301 cattgtagac ccacctactc gggtgaatat aaagctcgca cagatcatct cacagacatc
     3361 agaccttggc aaaggagcat cctgaacatc tctacacact gaggcaacga attcacctcg
     3421 agaccatggc tccaaagaag aagcgtaagg taaccatgag cgagctgatt aaggagaaca
     3481 tgcacatgaa gctgtacatg gagggcaccg tggacaacca tcacttcaag tgcacatccg
     3541 agggcgaagg caagccctac gagggcaccc agaccatgag aatcaaggtg gtcgagggcg
     3601 gccctctccc cttcgccttc gacatcctgg ctactagctt cctctacggc agcaagacct
     3661 tcatcaacca cacccagggc atccccgact tcttcaagca gtccttccct gagggcttca
     3721 catgggagag agtcaccaca tacgaagacg ggggcgtgct gaccgctacc caggacacca
     3781 gcctccagga cggctgcctc atctacaacg tcaagatcag aggggtgaac ttcacatcca
     3841 acggccctgt gatgcagaag aaaacactcg gctgggaggc cttcaccgag acgctgtacc
     3901 ccgctgacgg cggcctggaa ggcagaaacg acatggccct gaagctcgtg ggcgggagcc
     3961 atctgatcgc aaacatcaag accacatata gatccaagaa acccgctaag aacctcaaga
     4021 tgcctggcgt ctactatgtg gactacagac tggaaagaat caaggaggcc aacaacgaga
     4081 cctacgtcga gcagcacgag gtggcagtgg ccagatactg cgacctccct agcaaactgg
     4141 ggcacaagct caattaaagc tgactaattt atttagcagt agatagctat attgtgtgaa
     4201 gtggtaacag tccgtagatc tacggactgt taccacttca cgccgactca aactgcgcag
     4261 agaaacttca aacgacaaca ttggcatggc ttttgttatt tttggcgctt gactcaggat
     4321 ctaaaaactg gaacggtgaa ggtgacggca atgttttttg gcaaataagc atccccgaag
     4381 ttctacaatg catctgagga ctccatgttt tgttttcttt agtcattcca aatgtttgtt
     4441 aaatgcattg ttccgaaact tatttgcctc tatgaaggct gcccagtaat tgggagcata
     4501 cttaacattg tagtattgta tgtaaattat gtaacaaaac aatgtctggg tttttgtact
     4561 tttcagcctt aaaatcttgg gtttattttt ttttctttgt tccaaaaaaa cgaagcttta
     4621 ccattcaaga tgtaaaggtt tccattcccc ctgggcatat tgtaaaagct gtgtggaacg
     4681 tggcggtgcc agacatttgg tggggccaac ctgtacactg actaattcaa ttccaataaa
     4741 agtgcacatg taagacatcc tacccctgtc tcatttcctg tcttggctct gagtggcttc
     4801 aataaaaatg gcttttgtga agttgcacca tgttaattac atttcagcac tttcacacca
     4861 ggtttttgac taatttgaat ttatcgaagt ttgacctttt tttttttttt tcttcaagct
     4921 aatttgaggt tatcaaacca tttcattgga ggaaacttaa tgtttaaaag cataaattcg
     4981 aaacatgatt catttattta actgcttaat ggtgaaacaa acttcaagta atacaccaag
     5041 tgttccctca tatgaaggta gctaactgca aacggcctta actttccatc ttaagttgag
     5101 ctacataaag gctttaacag gaaggaactg ccagtttaaa agtcaaacca ccatgactga
     5161 tataaattct tgtccatata accttgcgtt tcacacaata tagctatcta ctgctaaata
     5221 aattagagct cataacttcg tataatgtat gctatacgaa gttatgttag ataacttcgt
     5281 ataaagtatc ctatacgaag ttatgcgagc taactttaaa taattggcat tatttaaagt
     5341 tatgaatcga taagtgaaga gctatccgga tatatatgta tgttatcata actgtactgg
     5401 ctatgtcagg tggtaatgac tgtaacgtta cgttactcgt tgtaggcacg acattgaata
     5461 tgctcttctc cgctgtggcc cgaacgcctc ccgctagcga tgggatctaa tacgactcac
     5521 tatagggaga ccaagggaaa atagaatgaa gtgatctcca aaaaataagt actttttgac
     5581 tgtaaataaa attgtaagga gtaaaaagta cttttttttc taaaaaaatg taattaagta
     5641 aaagtaaaag tattgatttt taattgtact caagtaaagt aaaaatcccc aaaaataata
     5701 cttaagtaca gtaatcaagt aaaattactc aagtacttta cacctctggg atccagctgc
     5761 atcatgaaca ataaaactgt ctgcttacat aaacagtaat acaaggggtg ttatgagcca
     5821 tattcaacgg gaaacgtcga ggccgcgatt aaattccaac atggatgctg atttatatgg
     5881 gtataaatgg gctcgcgata atgtcgggca atcaggtgcg acaatctatc gcttgtatgg
     5941 gaagcccgat gcgccagagt tgtttctgaa acatggcaaa ggtagcgttg ccaatgatgt
     6001 tacagatgag atggtcagac taaactggct gacggaattt atgcctcttc cgaccatcaa
     6061 gcattttatc cgtactcctg atgatgcatg gttactcacc actgcgatcc ccggaaaaac
     6121 agcattccag gtattagaag aatatcctga ttcaggtgaa aatattgttg atgcgctggc
     6181 agtgttcctg cgccggttgc attcgattcc tgtttgtaat tgtcctttta acagcgatcg
     6241 cgtatttcgt ctcgctcagg cgcaatcacg aatgaataac ggtttggttg atgcgagtga
     6301 ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc tggaaagaaa tgcataaact
     6361 tttgccattc tcaccggatt cagtcgtcac tcatggtgat ttctcacttg ataaccttat
     6421 ttttgacgag gggaaattaa taggttgtat tgatgttgga cgagtcggaa tcgcagaccg
     6481 ataccaggat cttgccatcc tatggaactg cctcggtgag ttttctcctt cattacagaa
     6541 acggcttttt caaaaatatg gtattgataa tcctgatatg aataaattgc agtttcattt
     6601 gatgctcgat gagtttttct aatcagaatt ggttaattgg ttgtaacact ggcaaaaagg
     6661 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     6721 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     6781 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     6841 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     6901 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     6961 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     7021 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     7081 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     7141 tacctacagc gtgagctatg agaaagcgcc acggttcccg aagggagaaa ggcggacagg
     7201 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     7261 gcctggtatc tttatagtcc tgt
//
