LOCUS       sequence-347617-        7558 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 7558)
  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 7558)
  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-; gcry1:BFP -0
FEATURES             Location/Qualifiers
     source          1..7558
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     24..43
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     277..294
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    join(324..335,336..498)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     protein_bind    585..606
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(621..626,627..644,645..651)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    659..675
                     /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     664..686
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     683..699
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     683..699
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             695..1006
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWHLPHFRPHNFK"
     promoter        720..738
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        complement(786..804)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(814..831)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(814..830)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(823..845)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     protein_bind    994..1025
                     /label=rox
                     /bound_moiety="Dre recombinase"
     protein_bind    complement(1031..1064)
                     /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(1069..1102)
                     /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             1247..1303
                     /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             1304..1744
                     /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             3760..3780
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3784..4485
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     protein_bind    5560..5593
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    5599..5632
                     /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    5639..5670
                     /label=rox
                     /bound_moiety="Dre recombinase"
     promoter        5836..5854
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(5877..6064,6065..6076)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(6130..6149)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             6141..6950
                     /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(6208..6227)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      join(7093..7558,1..123)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
       61 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
      121 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
      181 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
      241 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
      301 gaacagattt aaatggtacc gagcagaggt gtaaaaagta ctcaaaaatt ttactcaagt
      361 gaaagtacaa gtacttaggg aaaattttac tcaattaaaa gtaaaagtat ctggctagaa
      421 tcttacttga gtaaaagtaa aaaagtactc cattaaaatt gtacttgagt attaaggaag
      481 taaaagtaaa agcaagaaag aaaactagag tggtctccct ttagtgaggg tcaattgatg
      541 ggaggcgttc gggccacagc ggacacgcag gtgcgcaacg caattaatgt gagttagctc
      601 actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt
      661 gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc caagcgcgca
      721 attaaccctc actaaaggga acaaaagctg gaggacctcg agggggggcc cggtacccaa
      781 ttcgccctat agtgagtcgt attacgcgcg ctcactggcc gtcgttttac aacgtcgtga
      841 ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag
      901 ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa
      961 tggcgaatgg cacctgccac acttccggcc gcataacttt aaataatgcc aattatttaa
     1021 agttactatc ataacttcgt atagcataca ttatacgaag ttatcaccat aacttcgtat
     1081 aggatacttt atacgaagtt atcctaggct taccacccac tagcggtcag actgcagatt
     1141 gcagcacgaa acaggaagct gactccacat ggtcacatgc tcactgaagt gttgacttcc
     1201 ctgacagctg tgcactttct aaaccggttt tctcattcat ttacaggcca cgaacttctc
     1261 tctgttaaag caagcaggag acgtggaaga aaaccccggt cctatgaaac tgctctcatc
     1321 catcgagcaa gcctgcgaca tttgtcggct taagaagctg aaatgctcca aggaaaagcc
     1381 gaaatgtgcc aaatgcctga agaacaattg ggaatgtcgt tactctccca aaaccaagcg
     1441 aagtccactc acaagggctc atctgaccga agtggagagc aggctagaga gactggaaca
     1501 actctttttg ctcatcttcc ctagagagga ccttgacatg atcctcaaga tggattctct
     1561 ccaggatatt aaagcccttt tgactggctt attcgtccag gacaatgtga acaaagacgc
     1621 tgtgacagac cgattggcaa gtgtcgagac cgatatgcct ctgacactga gacagcacag
     1681 aatcagcgct acttcctcaa gcgaagagtc ttctaacaag ggacagagac agctgactgt
     1741 ttcgagcagg tcgaccccgt ccccggccga cgccctggac gacggcgacc tggacatgct
     1801 gcctgctgat gctctcgatg atttcgatct ggatatgctc ccggccgacg ccctggacga
     1861 ctacgacctg gacatcctcc cgggtaacta ataattaagt ctcagccacc gttaactgaa
     1921 catgtcaaaa cctgtggaga ctgttgagat ttgatgttct gaaaagataa agcctataaa
     1981 taaaatgttg cccaaatttc ctgcctgatg tttttctttg tctttgctac atggctttgc
     2041 tgctcggatc ggctcactct gtgtatgcca cgttcacttt gtactctcct tctcacggta
     2101 ggtttattat ttttagatgt gcagttagtt tctgtgaaat aacacaccac acactgatat
     2161 tgtctgtgca ttgacttggt gagtgcacat tgtttttgat cttgacatat ttatatttga
     2221 ttgatcaggt gaactgtgtg aatctaaagt gctccataca gatgttctgc attgaaaata
     2281 ttctcatttt attagtggaa gtgagtgtat gccacatcca atcaatttca gcaaacaccc
     2341 cagtatgatt taatgcaaaa aaatgaaggt atcaaacacg cattactact ttgcagttaa
     2401 atatttaaca tttattccaa cacgaaaaaa agcagtaaat aacactttga caaacacgtc
     2461 aggacatctt atttttgtca ccctcacagg caatttagta taatatatta tatatatata
     2521 tatatcatat aataatattc agtataatat atatatatat atcatattat aatattcagt
     2581 ataatataaa acacaaacac atatatgtat aatataatat aacattttta tttattgaga
     2641 tgcctctatg gaccgtgtta taagaagtaa agatcaggag aagtaaacat gaagtgtaat
     2701 tatgaatact gatgttaaat taagctatga tgagttttca ctgttaattt accatctcaa
     2761 ttaaatgttg atgcctccat gaccaagtta agcagatgag actgaggaca actgtagaag
     2821 acaagatgtt cactttgctg aatatagctg gcttgacagt tatctatgac tctataaata
     2881 tatatatatt ttttttttta taaaatgatt tatttataac tatatatcca tttctcagac
     2941 aggtgcttca tatccctcac tcccgtagct gtccatgctg gatctgtccc cgttgttttt
     3001 aaaaagctaa ataagttatt aacatgactg catccagcga gccaaacctg tctggtgtac
     3061 agctaccaga gaagcttgag atcctagtca ccggcgttcc tagttcttta aactgttaca
     3121 aagtgtttgg accagcaact gaattgcact gtaaattacc acatcagggt ctccggggtc
     3181 aacgctgcta agcaatggac tggaaaattt gtgacattga gtttttggaa taactgggaa
     3241 tgctggataa ctctttgggt aaagctatgg aacaaaagta ccagcatgaa ctaagaaatg
     3301 actgtcacag cacaacatca aacacaatgc aagacaaagg ttctctgctt ttgccttact
     3361 tgacagaaaa agtaagctct aggttcatcg catgcagaca gcagcaatac atgacatata
     3421 taaaggctgg ctcttttctc ccttccattg aactgaaact tccactcagt cagacttgcg
     3481 ggggatccac agcctgacta actcgacaaa cccccccaaa cctaaggtga gttgatcttt
     3541 aagcttttta cattttcagc tcgcatatat caattcgcgc gcattgtcac actagcaccc
     3601 actagcggtc agactgcaga ttgcagcacg aaacaggaag ctgactccac atggtcacat
     3661 gctcactgaa gtgttgactt ccctgacagc tgtgcacttt ctaaaccggt tttctcattc
     3721 atttacagtt cagccggaat tcacctcgag accatggctc caaagaagaa gcgtaaggta
     3781 accatgagcg agctgattaa ggagaacatg cacatgaagc tgtacatgga gggcaccgtg
     3841 gacaaccatc acttcaagtg cacatccgag ggcgaaggca agccctacga gggcacccag
     3901 accatgagaa tcaaggtggt cgagggcggc cctctcccct tcgccttcga catcctggct
     3961 actagcttcc tctacggcag caagaccttc atcaaccaca cccagggcat ccccgacttc
     4021 ttcaagcagt ccttccctga gggcttcaca tgggagagag tcaccacata cgaagacggg
     4081 ggcgtgctga ccgctaccca ggacaccagc ctccaggacg gctgcctcat ctacaacgtc
     4141 aagatcagag gggtgaactt cacatccaac ggccctgtga tgcagaagaa aacactcggc
     4201 tgggaggcct tcaccgagac gctgtacccc gctgacggcg gcctggaagg cagaaacgac
     4261 atggccctga agctcgtggg cgggagccat ctgatcgcaa acatcaagac cacatataga
     4321 tccaagaaac ccgctaagaa cctcaagatg cctggcgtct actatgtgga ctacagactg
     4381 gaaagaatca aggaggccaa caacgagacc tacgtcgagc agcacgaggt ggcagtggcc
     4441 agatactgcg acctccctag caaactgggg cacaagctca attaaagctg actaatttat
     4501 ttagcagtag atagctatat tgtgtgaagt ggtaacagtc cgtagatcta cggactgtta
     4561 ccacttcacg ccgactcaaa ctgcgcagag aaacttcaaa cgacaacatt ggcatggctt
     4621 ttgttatttt tggcgcttga ctcaggatct aaaaactgga acggtgaagg tgacggcaat
     4681 gttttttggc aaataagcat ccccgaagtt ctacaatgca tctgaggact ccatgttttg
     4741 ttttctttag tcattccaaa tgtttgttaa atgcattgtt ccgaaactta tttgcctcta
     4801 tgaaggctgc ccagtaattg ggagcatact taacattgta gtattgtatg taaattatgt
     4861 aacaaaacaa tgtctgggtt tttgtacttt tcagccttaa aatcttgggt ttattttttt
     4921 ttctttgttc caaaaaaacg aagctttacc attcaagatg taaaggtttc cattccccct
     4981 gggcatattg taaaagctgt gtggaacgtg gcggtgccag acatttggtg gggccaacct
     5041 gtacactgac taattcaatt ccaataaaag tgcacatgta agacatccta cccctgtctc
     5101 atttcctgtc ttggctctga gtggcttcaa taaaaatggc ttttgtgaag ttgcaccatg
     5161 ttaattacat ttcagcactt tcacaccagg tttttgacta atttgaattt atcgaagttt
     5221 gacctttttt tttttttttc ttcaagctaa tttgaggtta tcaaaccatt tcattggagg
     5281 aaacttaatg tttaaaagca taaattcgaa acatgattca tttatttaac tgcttaatgg
     5341 tgaaacaaac ttcaagtaat acaccaagtg ttccctcata tgaaggtagc taactgcaaa
     5401 cggccttaac tttccatctt aagttgagct acataaaggc tttaacagga aggaactgcc
     5461 agtttaaaag tcaaaccacc atgactgata taaattcttg tccatataac cttgcgtttc
     5521 acacaatata gctatctact gctaaataaa ttagagctca taacttcgta taatgtatgc
     5581 tatacgaagt tatgttagat aacttcgtat aaagtatcct atacgaagtt atgcgagcta
     5641 actttaaata attggcatta tttaaagtta tgaatcgata agtgaagagc tatccggata
     5701 tatatgtatg ttatcataac tgtactggct atgtcaggtg gtaatgactg taacgttacg
     5761 ttactcgttg taggcacgac attgaatatg ctcttctccg ctgtggcccg aacgcctccc
     5821 gctagcgatg ggatctaata cgactcacta tagggagacc aagggaaaat agaatgaagt
     5881 gatctccaaa aaataagtac tttttgactg taaataaaat tgtaaggagt aaaaagtact
     5941 tttttttcta aaaaaatgta attaagtaaa agtaaaagta ttgattttta attgtactca
     6001 agtaaagtaa aaatccccaa aaataatact taagtacagt aatcaagtaa aattactcaa
     6061 gtactttaca cctctgggat ccagctgcat catgaacaat aaaactgtct gcttacataa
     6121 acagtaatac aaggggtgtt atgagccata ttcaacggga aacgtcgagg ccgcgattaa
     6181 attccaacat ggatgctgat ttatatgggt ataaatgggc tcgcgataat gtcgggcaat
     6241 caggtgcgac aatctatcgc ttgtatggga agcccgatgc gccagagttg tttctgaaac
     6301 atggcaaagg tagcgttgcc aatgatgtta cagatgagat ggtcagacta aactggctga
     6361 cggaatttat gcctcttccg accatcaagc attttatccg tactcctgat gatgcatggt
     6421 tactcaccac tgcgatcccc ggaaaaacag cattccaggt attagaagaa tatcctgatt
     6481 caggtgaaaa tattgttgat gcgctggcag tgttcctgcg ccggttgcat tcgattcctg
     6541 tttgtaattg tccttttaac agcgatcgcg tatttcgtct cgctcaggcg caatcacgaa
     6601 tgaataacgg tttggttgat gcgagtgatt ttgatgacga gcgtaatggc tggcctgttg
     6661 aacaagtctg gaaagaaatg cataaacttt tgccattctc accggattca gtcgtcactc
     6721 atggtgattt ctcacttgat aaccttattt ttgacgaggg gaaattaata ggttgtattg
     6781 atgttggacg agtcggaatc gcagaccgat accaggatct tgccatccta tggaactgcc
     6841 tcggtgagtt ttctccttca ttacagaaac ggctttttca aaaatatggt attgataatc
     6901 ctgatatgaa taaattgcag tttcatttga tgctcgatga gtttttctaa tcagaattgg
     6961 ttaattggtt gtaacactgg caaaaaggat ctaggtgaag atcctttttg ataatctcat
     7021 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     7081 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     7141 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     7201 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     7261 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     7321 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     7381 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     7441 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     7501 ggttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacagga
//
