LOCUS       sequence-347618-        7560 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 7560)
  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 7560)
  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 -1
FEATURES             Location/Qualifiers
     source          1..7560
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     41..60
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     294..311
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    join(341..352,353..515)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     protein_bind    602..623
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(638..643,644..661,662..668)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    676..692
                     /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     681..703
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     700..716
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     700..716
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             712..1023
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWHLPHFRPHNFK"
     promoter        737..755
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        complement(803..821)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(831..848)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(831..847)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(840..862)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     protein_bind    1011..1042
                     /label=rox
                     /bound_moiety="Dre recombinase"
     protein_bind    complement(1048..1081)
                     /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(1086..1119)
                     /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             1266..1322
                     /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             1323..1763
                     /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             3779..3799
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3803..4504
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     protein_bind    5579..5612
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    5618..5651
                     /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    5658..5689
                     /label=rox
                     /bound_moiety="Dre recombinase"
     promoter        5855..5873
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(5896..6083,6084..6095)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(6149..6168)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             6160..6969
                     /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(6227..6246)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      join(7112..7560,1..140)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
       61 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
      121 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
      181 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
      241 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
      301 cagtgagcga ggaagcggaa cagatttaaa tggtaccgag cagaggtgta aaaagtactc
      361 aaaaatttta ctcaagtgaa agtacaagta cttagggaaa attttactca attaaaagta
      421 aaagtatctg gctagaatct tacttgagta aaagtaaaaa agtactccat taaaattgta
      481 cttgagtatt aaggaagtaa aagtaaaagc aagaaagaaa actagagtgg tctcccttta
      541 gtgagggtca attgatggga ggcgttcggg ccacagcgga cacgcaggtg cgcaacgcaa
      601 ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc
      661 gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg
      721 attacgccaa gcgcgcaatt aaccctcact aaagggaaca aaagctggag gacctcgagg
      781 gggggcccgg tacccaattc gccctatagt gagtcgtatt acgcgcgctc actggccgtc
      841 gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca
      901 catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa
      961 cagttgcgca gcctgaatgg cgaatggcac ctgccacact tccggccgca taactttaaa
     1021 taatgccaat tatttaaagt tactatcata acttcgtata gcatacatta tacgaagtta
     1081 tcaccataac ttcgtatagg atactttata cgaagttatc ctaggcttac cacccactag
     1141 cggtcagact gcagattgca gcacgaaaca ggaagctgac tccacatggt cacatgctca
     1201 ctgaagtgtt gacttccctg acagctgtgc actttctaaa ccggttttct cattcattta
     1261 cagttgccac gaacttctct ctgttaaagc aagcaggaga cgtggaagaa aaccccggtc
     1321 ctatgaaact gctctcatcc atcgagcaag cctgcgacat ttgtcggctt aagaagctga
     1381 aatgctccaa ggaaaagccg aaatgtgcca aatgcctgaa gaacaattgg gaatgtcgtt
     1441 actctcccaa aaccaagcga agtccactca caagggctca tctgaccgaa gtggagagca
     1501 ggctagagag actggaacaa ctctttttgc tcatcttccc tagagaggac cttgacatga
     1561 tcctcaagat ggattctctc caggatatta aagccctttt gactggctta ttcgtccagg
     1621 acaatgtgaa caaagacgct gtgacagacc gattggcaag tgtcgagacc gatatgcctc
     1681 tgacactgag acagcacaga atcagcgcta cttcctcaag cgaagagtct tctaacaagg
     1741 gacagagaca gctgactgtt tcgagcaggt cgaccccgtc cccggccgac gccctggacg
     1801 acggcgacct ggacatgctg cctgctgatg ctctcgatga tttcgatctg gatatgctcc
     1861 cggccgacgc cctggacgac tacgacctgg acatcctccc gggtaactaa taattaagtc
     1921 tcagccaccg ttaactgaac atgtcaaaac ctgtggagac tgttgagatt tgatgttctg
     1981 aaaagataaa gcctataaat aaaatgttgc ccaaatttcc tgcctgatgt ttttctttgt
     2041 ctttgctaca tggctttgct gctcggatcg gctcactctg tgtatgccac gttcactttg
     2101 tactctcctt ctcacggtag gtttattatt tttagatgtg cagttagttt ctgtgaaata
     2161 acacaccaca cactgatatt gtctgtgcat tgacttggtg agtgcacatt gtttttgatc
     2221 ttgacatatt tatatttgat tgatcaggtg aactgtgtga atctaaagtg ctccatacag
     2281 atgttctgca ttgaaaatat tctcatttta ttagtggaag tgagtgtatg ccacatccaa
     2341 tcaatttcag caaacacccc agtatgattt aatgcaaaaa aatgaaggta tcaaacacgc
     2401 attactactt tgcagttaaa tatttaacat ttattccaac acgaaaaaaa gcagtaaata
     2461 acactttgac aaacacgtca ggacatctta tttttgtcac cctcacaggc aatttagtat
     2521 aatatattat atatatatat atatcatata ataatattca gtataatata tatatatata
     2581 tcatattata atattcagta taatataaaa cacaaacaca tatatgtata atataatata
     2641 acatttttat ttattgagat gcctctatgg accgtgttat aagaagtaaa gatcaggaga
     2701 agtaaacatg aagtgtaatt atgaatactg atgttaaatt aagctatgat gagttttcac
     2761 tgttaattta ccatctcaat taaatgttga tgcctccatg accaagttaa gcagatgaga
     2821 ctgaggacaa ctgtagaaga caagatgttc actttgctga atatagctgg cttgacagtt
     2881 atctatgact ctataaatat atatatattt ttttttttat aaaatgattt atttataact
     2941 atatatccat ttctcagaca ggtgcttcat atccctcact cccgtagctg tccatgctgg
     3001 atctgtcccc gttgttttta aaaagctaaa taagttatta acatgactgc atccagcgag
     3061 ccaaacctgt ctggtgtaca gctaccagag aagcttgaga tcctagtcac cggcgttcct
     3121 agttctttaa actgttacaa agtgtttgga ccagcaactg aattgcactg taaattacca
     3181 catcagggtc tccggggtca acgctgctaa gcaatggact ggaaaatttg tgacattgag
     3241 tttttggaat aactgggaat gctggataac tctttgggta aagctatgga acaaaagtac
     3301 cagcatgaac taagaaatga ctgtcacagc acaacatcaa acacaatgca agacaaaggt
     3361 tctctgcttt tgccttactt gacagaaaaa gtaagctcta ggttcatcgc atgcagacag
     3421 cagcaataca tgacatatat aaaggctggc tcttttctcc cttccattga actgaaactt
     3481 ccactcagtc agacttgcgg gggatccaca gcctgactaa ctcgacaaac ccccccaaac
     3541 ctaaggtgag ttgatcttta agctttttac attttcagct cgcatatatc aattcgcgcg
     3601 cattgtcaca ctagcaccca ctagcggtca gactgcagat tgcagcacga aacaggaagc
     3661 tgactccaca tggtcacatg ctcactgaag tgttgacttc cctgacagct gtgcactttc
     3721 taaaccggtt ttctcattca tttacagttc agccggaatt cacctcgaga ccatggctcc
     3781 aaagaagaag cgtaaggtaa ccatgagcga gctgattaag gagaacatgc acatgaagct
     3841 gtacatggag ggcaccgtgg acaaccatca cttcaagtgc acatccgagg gcgaaggcaa
     3901 gccctacgag ggcacccaga ccatgagaat caaggtggtc gagggcggcc ctctcccctt
     3961 cgccttcgac atcctggcta ctagcttcct ctacggcagc aagaccttca tcaaccacac
     4021 ccagggcatc cccgacttct tcaagcagtc cttccctgag ggcttcacat gggagagagt
     4081 caccacatac gaagacgggg gcgtgctgac cgctacccag gacaccagcc tccaggacgg
     4141 ctgcctcatc tacaacgtca agatcagagg ggtgaacttc acatccaacg gccctgtgat
     4201 gcagaagaaa acactcggct gggaggcctt caccgagacg ctgtaccccg ctgacggcgg
     4261 cctggaaggc agaaacgaca tggccctgaa gctcgtgggc gggagccatc tgatcgcaaa
     4321 catcaagacc acatatagat ccaagaaacc cgctaagaac ctcaagatgc ctggcgtcta
     4381 ctatgtggac tacagactgg aaagaatcaa ggaggccaac aacgagacct acgtcgagca
     4441 gcacgaggtg gcagtggcca gatactgcga cctccctagc aaactggggc acaagctcaa
     4501 ttaaagctga ctaatttatt tagcagtaga tagctatatt gtgtgaagtg gtaacagtcc
     4561 gtagatctac ggactgttac cacttcacgc cgactcaaac tgcgcagaga aacttcaaac
     4621 gacaacattg gcatggcttt tgttattttt ggcgcttgac tcaggatcta aaaactggaa
     4681 cggtgaaggt gacggcaatg ttttttggca aataagcatc cccgaagttc tacaatgcat
     4741 ctgaggactc catgttttgt tttctttagt cattccaaat gtttgttaaa tgcattgttc
     4801 cgaaacttat ttgcctctat gaaggctgcc cagtaattgg gagcatactt aacattgtag
     4861 tattgtatgt aaattatgta acaaaacaat gtctgggttt ttgtactttt cagccttaaa
     4921 atcttgggtt tatttttttt tctttgttcc aaaaaaacga agctttacca ttcaagatgt
     4981 aaaggtttcc attccccctg ggcatattgt aaaagctgtg tggaacgtgg cggtgccaga
     5041 catttggtgg ggccaacctg tacactgact aattcaattc caataaaagt gcacatgtaa
     5101 gacatcctac ccctgtctca tttcctgtct tggctctgag tggcttcaat aaaaatggct
     5161 tttgtgaagt tgcaccatgt taattacatt tcagcacttt cacaccaggt ttttgactaa
     5221 tttgaattta tcgaagtttg accttttttt ttttttttct tcaagctaat ttgaggttat
     5281 caaaccattt cattggagga aacttaatgt ttaaaagcat aaattcgaaa catgattcat
     5341 ttatttaact gcttaatggt gaaacaaact tcaagtaata caccaagtgt tccctcatat
     5401 gaaggtagct aactgcaaac ggccttaact ttccatctta agttgagcta cataaaggct
     5461 ttaacaggaa ggaactgcca gtttaaaagt caaaccacca tgactgatat aaattcttgt
     5521 ccatataacc ttgcgtttca cacaatatag ctatctactg ctaaataaat tagagctcat
     5581 aacttcgtat aatgtatgct atacgaagtt atgttagata acttcgtata aagtatccta
     5641 tacgaagtta tgcgagctaa ctttaaataa ttggcattat ttaaagttat gaatcgataa
     5701 gtgaagagct atccggatat atatgtatgt tatcataact gtactggcta tgtcaggtgg
     5761 taatgactgt aacgttacgt tactcgttgt aggcacgaca ttgaatatgc tcttctccgc
     5821 tgtggcccga acgcctcccg ctagcgatgg gatctaatac gactcactat agggagacca
     5881 agggaaaata gaatgaagtg atctccaaaa aataagtact ttttgactgt aaataaaatt
     5941 gtaaggagta aaaagtactt ttttttctaa aaaaatgtaa ttaagtaaaa gtaaaagtat
     6001 tgatttttaa ttgtactcaa gtaaagtaaa aatccccaaa aataatactt aagtacagta
     6061 atcaagtaaa attactcaag tactttacac ctctgggatc cagctgcatc atgaacaata
     6121 aaactgtctg cttacataaa cagtaataca aggggtgtta tgagccatat tcaacgggaa
     6181 acgtcgaggc cgcgattaaa ttccaacatg gatgctgatt tatatgggta taaatgggct
     6241 cgcgataatg tcgggcaatc aggtgcgaca atctatcgct tgtatgggaa gcccgatgcg
     6301 ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca atgatgttac agatgagatg
     6361 gtcagactaa actggctgac ggaatttatg cctcttccga ccatcaagca ttttatccgt
     6421 actcctgatg atgcatggtt actcaccact gcgatccccg gaaaaacagc attccaggta
     6481 ttagaagaat atcctgattc aggtgaaaat attgttgatg cgctggcagt gttcctgcgc
     6541 cggttgcatt cgattcctgt ttgtaattgt ccttttaaca gcgatcgcgt atttcgtctc
     6601 gctcaggcgc aatcacgaat gaataacggt ttggttgatg cgagtgattt tgatgacgag
     6661 cgtaatggct ggcctgttga acaagtctgg aaagaaatgc ataaactttt gccattctca
     6721 ccggattcag tcgtcactca tggtgatttc tcacttgata accttatttt tgacgagggg
     6781 aaattaatag gttgtattga tgttggacga gtcggaatcg cagaccgata ccaggatctt
     6841 gccatcctat ggaactgcct cggtgagttt tctccttcat tacagaaacg gctttttcaa
     6901 aaatatggta ttgataatcc tgatatgaat aaattgcagt ttcatttgat gctcgatgag
     6961 tttttctaat cagaattggt taattggttg taacactggc aaaaaggatc taggtgaaga
     7021 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     7081 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     7141 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     7201 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     7261 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     7321 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     7381 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     7441 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     7501 agctatgaga aagcgccacg gttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
//
