LOCUS       sequence-345368-        7559 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 7559)
  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 7559)
  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 -2
FEATURES             Location/Qualifiers
     source          1..7559
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     252..269
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    join(299..310,311..473)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     protein_bind    560..581
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(596..601,602..619,620..626)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    634..650
                     /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     639..661
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     658..674
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     658..674
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             670..981
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWHLPHFRPHNFK"
     promoter        695..713
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        complement(761..779)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(789..806)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(789..805)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(798..820)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     protein_bind    969..1000
                     /label=rox
                     /bound_moiety="Dre recombinase"
     protein_bind    complement(1006..1039)
                     /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(1044..1077)
                     /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             1223..1279
                     /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             1280..1720
                     /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             3736..3756
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3760..4461
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     protein_bind    5536..5569
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    5575..5608
                     /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    5615..5646
                     /label=rox
                     /bound_moiety="Dre recombinase"
     promoter        5812..5830
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(5853..6040,6041..6052)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(6106..6125)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             6117..6926
                     /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(6184..6203)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      join(7069..7559,1..98)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     join(7558..7559,1..18)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
       61 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
      121 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
      181 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
      241 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaca gatttaaatg gtaccgagca
      301 gaggtgtaaa aagtactcaa aaattttact caagtgaaag tacaagtact tagggaaaat
      361 tttactcaat taaaagtaaa agtatctggc tagaatctta cttgagtaaa agtaaaaaag
      421 tactccatta aaattgtact tgagtattaa ggaagtaaaa gtaaaagcaa gaaagaaaac
      481 tagagtggtc tccctttagt gagggtcaat tgatgggagg cgttcgggcc acagcggaca
      541 cgcaggtgcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc caggctttac
      601 actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa tttcacacag
      661 gaaacagcta tgaccatgat tacgccaagc gcgcaattaa ccctcactaa agggaacaaa
      721 agctggagga cctcgagggg gggcccggta cccaattcgc cctatagtga gtcgtattac
      781 gcgcgctcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa
      841 cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc
      901 accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggcacct gccacacttc
      961 cggccgcata actttaaata atgccaatta tttaaagtta ctatcataac ttcgtatagc
     1021 atacattata cgaagttatc accataactt cgtataggat actttatacg aagttatcct
     1081 aggcttacca cccactagcg gtcagactgc agattgcagc acgaaacagg aagctgactc
     1141 cacatggtca catgctcact gaagtgttga cttccctgac agctgtgcac tttctaaacc
     1201 ggttttctca ttcatttaca gcgccacgaa cttctctctg ttaaagcaag caggagacgt
     1261 ggaagaaaac cccggtccta tgaaactgct ctcatccatc gagcaagcct gcgacatttg
     1321 tcggcttaag aagctgaaat gctccaagga aaagccgaaa tgtgccaaat gcctgaagaa
     1381 caattgggaa tgtcgttact ctcccaaaac caagcgaagt ccactcacaa gggctcatct
     1441 gaccgaagtg gagagcaggc tagagagact ggaacaactc tttttgctca tcttccctag
     1501 agaggacctt gacatgatcc tcaagatgga ttctctccag gatattaaag cccttttgac
     1561 tggcttattc gtccaggaca atgtgaacaa agacgctgtg acagaccgat tggcaagtgt
     1621 cgagaccgat atgcctctga cactgagaca gcacagaatc agcgctactt cctcaagcga
     1681 agagtcttct aacaagggac agagacagct gactgtttcg agcaggtcga ccccgtcccc
     1741 ggccgacgcc ctggacgacg gcgacctgga catgctgcct gctgatgctc tcgatgattt
     1801 cgatctggat atgctcccgg ccgacgccct ggacgactac gacctggaca tcctcccggg
     1861 taactaataa ttaagtctca gccaccgtta actgaacatg tcaaaacctg tggagactgt
     1921 tgagatttga tgttctgaaa agataaagcc tataaataaa atgttgccca aatttcctgc
     1981 ctgatgtttt tctttgtctt tgctacatgg ctttgctgct cggatcggct cactctgtgt
     2041 atgccacgtt cactttgtac tctccttctc acggtaggtt tattattttt agatgtgcag
     2101 ttagtttctg tgaaataaca caccacacac tgatattgtc tgtgcattga cttggtgagt
     2161 gcacattgtt tttgatcttg acatatttat atttgattga tcaggtgaac tgtgtgaatc
     2221 taaagtgctc catacagatg ttctgcattg aaaatattct cattttatta gtggaagtga
     2281 gtgtatgcca catccaatca atttcagcaa acaccccagt atgatttaat gcaaaaaaat
     2341 gaaggtatca aacacgcatt actactttgc agttaaatat ttaacattta ttccaacacg
     2401 aaaaaaagca gtaaataaca ctttgacaaa cacgtcagga catcttattt ttgtcaccct
     2461 cacaggcaat ttagtataat atattatata tatatatata tcatataata atattcagta
     2521 taatatatat atatatatca tattataata ttcagtataa tataaaacac aaacacatat
     2581 atgtataata taatataaca tttttattta ttgagatgcc tctatggacc gtgttataag
     2641 aagtaaagat caggagaagt aaacatgaag tgtaattatg aatactgatg ttaaattaag
     2701 ctatgatgag ttttcactgt taatttacca tctcaattaa atgttgatgc ctccatgacc
     2761 aagttaagca gatgagactg aggacaactg tagaagacaa gatgttcact ttgctgaata
     2821 tagctggctt gacagttatc tatgactcta taaatatata tatatttttt tttttataaa
     2881 atgatttatt tataactata tatccatttc tcagacaggt gcttcatatc cctcactccc
     2941 gtagctgtcc atgctggatc tgtccccgtt gtttttaaaa agctaaataa gttattaaca
     3001 tgactgcatc cagcgagcca aacctgtctg gtgtacagct accagagaag cttgagatcc
     3061 tagtcaccgg cgttcctagt tctttaaact gttacaaagt gtttggacca gcaactgaat
     3121 tgcactgtaa attaccacat cagggtctcc ggggtcaacg ctgctaagca atggactgga
     3181 aaatttgtga cattgagttt ttggaataac tgggaatgct ggataactct ttgggtaaag
     3241 ctatggaaca aaagtaccag catgaactaa gaaatgactg tcacagcaca acatcaaaca
     3301 caatgcaaga caaaggttct ctgcttttgc cttacttgac agaaaaagta agctctaggt
     3361 tcatcgcatg cagacagcag caatacatga catatataaa ggctggctct tttctccctt
     3421 ccattgaact gaaacttcca ctcagtcaga cttgcggggg atccacagcc tgactaactc
     3481 gacaaacccc cccaaaccta aggtgagttg atctttaagc tttttacatt ttcagctcgc
     3541 atatatcaat tcgcgcgcat tgtcacacta gcacccacta gcggtcagac tgcagattgc
     3601 agcacgaaac aggaagctga ctccacatgg tcacatgctc actgaagtgt tgacttccct
     3661 gacagctgtg cactttctaa accggttttc tcattcattt acagttcagc cggaattcac
     3721 ctcgagacca tggctccaaa gaagaagcgt aaggtaacca tgagcgagct gattaaggag
     3781 aacatgcaca tgaagctgta catggagggc accgtggaca accatcactt caagtgcaca
     3841 tccgagggcg aaggcaagcc ctacgagggc acccagacca tgagaatcaa ggtggtcgag
     3901 ggcggccctc tccccttcgc cttcgacatc ctggctacta gcttcctcta cggcagcaag
     3961 accttcatca accacaccca gggcatcccc gacttcttca agcagtcctt ccctgagggc
     4021 ttcacatggg agagagtcac cacatacgaa gacgggggcg tgctgaccgc tacccaggac
     4081 accagcctcc aggacggctg cctcatctac aacgtcaaga tcagaggggt gaacttcaca
     4141 tccaacggcc ctgtgatgca gaagaaaaca ctcggctggg aggccttcac cgagacgctg
     4201 taccccgctg acggcggcct ggaaggcaga aacgacatgg ccctgaagct cgtgggcggg
     4261 agccatctga tcgcaaacat caagaccaca tatagatcca agaaacccgc taagaacctc
     4321 aagatgcctg gcgtctacta tgtggactac agactggaaa gaatcaagga ggccaacaac
     4381 gagacctacg tcgagcagca cgaggtggca gtggccagat actgcgacct ccctagcaaa
     4441 ctggggcaca agctcaatta aagctgacta atttatttag cagtagatag ctatattgtg
     4501 tgaagtggta acagtccgta gatctacgga ctgttaccac ttcacgccga ctcaaactgc
     4561 gcagagaaac ttcaaacgac aacattggca tggcttttgt tatttttggc gcttgactca
     4621 ggatctaaaa actggaacgg tgaaggtgac ggcaatgttt tttggcaaat aagcatcccc
     4681 gaagttctac aatgcatctg aggactccat gttttgtttt ctttagtcat tccaaatgtt
     4741 tgttaaatgc attgttccga aacttatttg cctctatgaa ggctgcccag taattgggag
     4801 catacttaac attgtagtat tgtatgtaaa ttatgtaaca aaacaatgtc tgggtttttg
     4861 tacttttcag ccttaaaatc ttgggtttat ttttttttct ttgttccaaa aaaacgaagc
     4921 tttaccattc aagatgtaaa ggtttccatt ccccctgggc atattgtaaa agctgtgtgg
     4981 aacgtggcgg tgccagacat ttggtggggc caacctgtac actgactaat tcaattccaa
     5041 taaaagtgca catgtaagac atcctacccc tgtctcattt cctgtcttgg ctctgagtgg
     5101 cttcaataaa aatggctttt gtgaagttgc accatgttaa ttacatttca gcactttcac
     5161 accaggtttt tgactaattt gaatttatcg aagtttgacc tttttttttt tttttcttca
     5221 agctaatttg aggttatcaa accatttcat tggaggaaac ttaatgttta aaagcataaa
     5281 ttcgaaacat gattcattta tttaactgct taatggtgaa acaaacttca agtaatacac
     5341 caagtgttcc ctcatatgaa ggtagctaac tgcaaacggc cttaactttc catcttaagt
     5401 tgagctacat aaaggcttta acaggaagga actgccagtt taaaagtcaa accaccatga
     5461 ctgatataaa ttcttgtcca tataaccttg cgtttcacac aatatagcta tctactgcta
     5521 aataaattag agctcataac ttcgtataat gtatgctata cgaagttatg ttagataact
     5581 tcgtataaag tatcctatac gaagttatgc gagctaactt taaataattg gcattattta
     5641 aagttatgaa tcgataagtg aagagctatc cggatatata tgtatgttat cataactgta
     5701 ctggctatgt caggtggtaa tgactgtaac gttacgttac tcgttgtagg cacgacattg
     5761 aatatgctct tctccgctgt ggcccgaacg cctcccgcta gcgatgggat ctaatacgac
     5821 tcactatagg gagaccaagg gaaaatagaa tgaagtgatc tccaaaaaat aagtactttt
     5881 tgactgtaaa taaaattgta aggagtaaaa agtacttttt tttctaaaaa aatgtaatta
     5941 agtaaaagta aaagtattga tttttaattg tactcaagta aagtaaaaat ccccaaaaat
     6001 aatacttaag tacagtaatc aagtaaaatt actcaagtac tttacacctc tgggatccag
     6061 ctgcatcatg aacaataaaa ctgtctgctt acataaacag taatacaagg ggtgttatga
     6121 gccatattca acgggaaacg tcgaggccgc gattaaattc caacatggat gctgatttat
     6181 atgggtataa atgggctcgc gataatgtcg ggcaatcagg tgcgacaatc tatcgcttgt
     6241 atgggaagcc cgatgcgcca gagttgtttc tgaaacatgg caaaggtagc gttgccaatg
     6301 atgttacaga tgagatggtc agactaaact ggctgacgga atttatgcct cttccgacca
     6361 tcaagcattt tatccgtact cctgatgatg catggttact caccactgcg atccccggaa
     6421 aaacagcatt ccaggtatta gaagaatatc ctgattcagg tgaaaatatt gttgatgcgc
     6481 tggcagtgtt cctgcgccgg ttgcattcga ttcctgtttg taattgtcct tttaacagcg
     6541 atcgcgtatt tcgtctcgct caggcgcaat cacgaatgaa taacggtttg gttgatgcga
     6601 gtgattttga tgacgagcgt aatggctggc ctgttgaaca agtctggaaa gaaatgcata
     6661 aacttttgcc attctcaccg gattcagtcg tcactcatgg tgatttctca cttgataacc
     6721 ttatttttga cgaggggaaa ttaataggtt gtattgatgt tggacgagtc ggaatcgcag
     6781 accgatacca ggatcttgcc atcctatgga actgcctcgg tgagttttct ccttcattac
     6841 agaaacggct ttttcaaaaa tatggtattg ataatcctga tatgaataaa ttgcagtttc
     6901 atttgatgct cgatgagttt ttctaatcag aattggttaa ttggttgtaa cactggcaaa
     6961 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     7021 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     7081 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     7141 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     7201 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     7261 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     7321 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     7381 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     7441 gagataccta cagcgtgagc tatgagaaag cgccacggtt cccgaaggga gaaaggcgga
     7501 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccagggg
//
