LOCUS       sequence-353835-        7284 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 7284)
  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 7284)
  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 -2
FEATURES             Location/Qualifiers
     source          1..7284
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     206..223
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    join(253..264,265..427)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     protein_bind    514..535
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(550..555,556..573,574..580)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    588..604
                     /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     593..615
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     612..628
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     612..628
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             624..935
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWHLPHFRPHNFK"
     promoter        649..667
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        complement(715..733)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(743..760)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(743..759)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(752..774)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     protein_bind    923..954
                     /label=rox
                     /bound_moiety="Dre recombinase"
     protein_bind    complement(960..993)
                     /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(998..1031)
                     /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             1177..1233
                     /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             1234..1674
                     /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             3415..3435
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3439..4140
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     protein_bind    5215..5248
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    5254..5287
                     /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    5294..5325
                     /label=rox
                     /bound_moiety="Dre recombinase"
     promoter        5491..5509
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(5532..5719,5720..5731)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(5785..5804)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5796..6605
                     /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(5863..5882)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      join(6748..7284,1..52)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7237..7256
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
       61 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
      121 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
      181 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aacagattta
      241 aatggtaccg agcagaggtg taaaaagtac tcaaaaattt tactcaagtg aaagtacaag
      301 tacttaggga aaattttact caattaaaag taaaagtatc tggctagaat cttacttgag
      361 taaaagtaaa aaagtactcc attaaaattg tacttgagta ttaaggaagt aaaagtaaaa
      421 gcaagaaaga aaactagagt ggtctccctt tagtgagggt caattgatgg gaggcgttcg
      481 ggccacagcg gacacgcagg tgcgcaacgc aattaatgtg agttagctca ctcattaggc
      541 accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata
      601 acaatttcac acaggaaaca gctatgacca tgattacgcc aagcgcgcaa ttaaccctca
      661 ctaaagggaa caaaagctgg aggacctcga gggggggccc ggtacccaat tcgccctata
      721 gtgagtcgta ttacgcgcgc tcactggccg tcgttttaca acgtcgtgac tgggaaaacc
      781 ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata
      841 gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggc
      901 acctgccaca cttccggccg cataacttta aataatgcca attatttaaa gttactatca
      961 taacttcgta tagcatacat tatacgaagt tatcaccata acttcgtata ggatacttta
     1021 tacgaagtta tcctaggctt accacccact agcggtcaga ctgcagattg cagcacgaaa
     1081 caggaagctg actccacatg gtcacatgct cactgaagtg ttgacttccc tgacagctgt
     1141 gcactttcta aaccggtttt ctcattcatt tacagcgcca cgaacttctc tctgttaaag
     1201 caagcaggag acgtggaaga aaaccccggt cctatgaaac tgctctcatc catcgagcaa
     1261 gcctgcgaca tttgtcggct taagaagctg aaatgctcca aggaaaagcc gaaatgtgcc
     1321 aaatgcctga agaacaattg ggaatgtcgt tactctccca aaaccaagcg aagtccactc
     1381 acaagggctc atctgaccga agtggagagc aggctagaga gactggaaca actctttttg
     1441 ctcatcttcc ctagagagga ccttgacatg atcctcaaga tggattctct ccaggatatt
     1501 aaagcccttt tgactggctt attcgtccag gacaatgtga acaaagacgc tgtgacagac
     1561 cgattggcaa gtgtcgagac cgatatgcct ctgacactga gacagcacag aatcagcgct
     1621 acttcctcaa gcgaagagtc ttctaacaag ggacagagac agctgactgt ttcgagcagg
     1681 tcgaccccgt ccccggccga cgccctggac gacggcgacc tggacatgct gcctgctgat
     1741 gctctcgatg atttcgatct ggatatgctc ccggccgacg ccctggacga ctacgacctg
     1801 gacatcctcc cgggtaacta ataattaagt ctcagccacc gttaactgaa catgtcaaaa
     1861 cctgtggaga ctgttgagat ttgatgttct gaaaagataa agcctataaa taaaatgttg
     1921 cccaaatttc ctgcctgatg tttttctttg tctttgctac atggctttgc tgctcggatc
     1981 ggctcactct gtgtatgcca cgttcacttt gtactctcct tctcacggta ggtttattat
     2041 ttttagatgt gcagttagtt tctgtgaaat aacacaccac acactgatat tgtctgtgca
     2101 ttgacttggt gagtgcacat tgtttttgat cttgacatat ttatatttga ttgatcaggt
     2161 gaactgtgtg aatctaaagt gctccataca gatgttctgc attgaaaata ttctcatttt
     2221 attagtggaa gtgagtgtat gccacatcca atcaatttca gcaaacaccc cagtatgatt
     2281 taatgcaaaa aaatgaaggt atcaaacacg cattactact ttgcagttaa atatttaaca
     2341 tttattccaa cacgaaaaaa agcagtaaat aacactttga caaacacgtc aggacatctt
     2401 atttttgtca ccctcacagg caatttagta taatatatta tatatatata tatatcatat
     2461 aataatattc agtataatat atatatatat atcatattat aatattcagt ataatataaa
     2521 acacaaacac atatatgtat aatataatat aacattttta tttattgaga tgcctctatg
     2581 gaccgtgtta taagaagtaa agatcaggag aagtaaacat gaagtgtaat tatgaatact
     2641 gatgttaaat taagctatga tgagttttca ctgttaattt accatctcaa ttaaatgttg
     2701 atgcctccat gaccaagtta agcagatgag actgaggaca actgtagaag acaagatgtt
     2761 cactttgctg aatatagctg gcttgacagt tatctatgac tctataaata tatatatatt
     2821 ttttttttta taaaatgatt tatttataac tatatatcca tttctcagac aggtgcttca
     2881 tatccctcac tcccgtagct gtccatgctg gatctgtccc cgttgttttt aaaaagctaa
     2941 ataagttatt aacatgactg catccagcga gccaaacctg tctggtgtac agctaccaga
     3001 gaagcttgag atcctagtca aagctttcaa ccactccagg catagctaga gccactgatg
     3061 catctataga gctgaagtaa agatagggtt gatgcaaatc gatttagaat tttcaatgta
     3121 gaaaaaacaa aataaaaagt ctattcactt gatggataga ttagtcaaac aaacatagag
     3181 taggcacttc ccttgactct gtaaactttt ggatttaaca gattgtgcac tccttttagt
     3241 taaacacatt agtccctgaa aactaagacc ctaaccataa tcccattgta gacccaccta
     3301 ctcgggtgaa tataaagctc gcacagatca tctcacagac atcagacctt ggcaaaggag
     3361 catcctgaac atctctacac actgaggcaa cgaattcacc tcgagaccat ggctccaaag
     3421 aagaagcgta aggtaaccat gagcgagctg attaaggaga acatgcacat gaagctgtac
     3481 atggagggca ccgtggacaa ccatcacttc aagtgcacat ccgagggcga aggcaagccc
     3541 tacgagggca cccagaccat gagaatcaag gtggtcgagg gcggccctct ccccttcgcc
     3601 ttcgacatcc tggctactag cttcctctac ggcagcaaga ccttcatcaa ccacacccag
     3661 ggcatccccg acttcttcaa gcagtccttc cctgagggct tcacatggga gagagtcacc
     3721 acatacgaag acgggggcgt gctgaccgct acccaggaca ccagcctcca ggacggctgc
     3781 ctcatctaca acgtcaagat cagaggggtg aacttcacat ccaacggccc tgtgatgcag
     3841 aagaaaacac tcggctggga ggccttcacc gagacgctgt accccgctga cggcggcctg
     3901 gaaggcagaa acgacatggc cctgaagctc gtgggcggga gccatctgat cgcaaacatc
     3961 aagaccacat atagatccaa gaaacccgct aagaacctca agatgcctgg cgtctactat
     4021 gtggactaca gactggaaag aatcaaggag gccaacaacg agacctacgt cgagcagcac
     4081 gaggtggcag tggccagata ctgcgacctc cctagcaaac tggggcacaa gctcaattaa
     4141 agctgactaa tttatttagc agtagatagc tatattgtgt gaagtggtaa cagtccgtag
     4201 atctacggac tgttaccact tcacgccgac tcaaactgcg cagagaaact tcaaacgaca
     4261 acattggcat ggcttttgtt atttttggcg cttgactcag gatctaaaaa ctggaacggt
     4321 gaaggtgacg gcaatgtttt ttggcaaata agcatccccg aagttctaca atgcatctga
     4381 ggactccatg ttttgttttc tttagtcatt ccaaatgttt gttaaatgca ttgttccgaa
     4441 acttatttgc ctctatgaag gctgcccagt aattgggagc atacttaaca ttgtagtatt
     4501 gtatgtaaat tatgtaacaa aacaatgtct gggtttttgt acttttcagc cttaaaatct
     4561 tgggtttatt tttttttctt tgttccaaaa aaacgaagct ttaccattca agatgtaaag
     4621 gtttccattc cccctgggca tattgtaaaa gctgtgtgga acgtggcggt gccagacatt
     4681 tggtggggcc aacctgtaca ctgactaatt caattccaat aaaagtgcac atgtaagaca
     4741 tcctacccct gtctcatttc ctgtcttggc tctgagtggc ttcaataaaa atggcttttg
     4801 tgaagttgca ccatgttaat tacatttcag cactttcaca ccaggttttt gactaatttg
     4861 aatttatcga agtttgacct tttttttttt ttttcttcaa gctaatttga ggttatcaaa
     4921 ccatttcatt ggaggaaact taatgtttaa aagcataaat tcgaaacatg attcatttat
     4981 ttaactgctt aatggtgaaa caaacttcaa gtaatacacc aagtgttccc tcatatgaag
     5041 gtagctaact gcaaacggcc ttaactttcc atcttaagtt gagctacata aaggctttaa
     5101 caggaaggaa ctgccagttt aaaagtcaaa ccaccatgac tgatataaat tcttgtccat
     5161 ataaccttgc gtttcacaca atatagctat ctactgctaa ataaattaga gctcataact
     5221 tcgtataatg tatgctatac gaagttatgt tagataactt cgtataaagt atcctatacg
     5281 aagttatgcg agctaacttt aaataattgg cattatttaa agttatgaat cgataagtga
     5341 agagctatcc ggatatatat gtatgttatc ataactgtac tggctatgtc aggtggtaat
     5401 gactgtaacg ttacgttact cgttgtaggc acgacattga atatgctctt ctccgctgtg
     5461 gcccgaacgc ctcccgctag cgatgggatc taatacgact cactataggg agaccaaggg
     5521 aaaatagaat gaagtgatct ccaaaaaata agtacttttt gactgtaaat aaaattgtaa
     5581 ggagtaaaaa gtactttttt ttctaaaaaa atgtaattaa gtaaaagtaa aagtattgat
     5641 ttttaattgt actcaagtaa agtaaaaatc cccaaaaata atacttaagt acagtaatca
     5701 agtaaaatta ctcaagtact ttacacctct gggatccagc tgcatcatga acaataaaac
     5761 tgtctgctta cataaacagt aatacaaggg gtgttatgag ccatattcaa cgggaaacgt
     5821 cgaggccgcg attaaattcc aacatggatg ctgatttata tgggtataaa tgggctcgcg
     5881 ataatgtcgg gcaatcaggt gcgacaatct atcgcttgta tgggaagccc gatgcgccag
     5941 agttgtttct gaaacatggc aaaggtagcg ttgccaatga tgttacagat gagatggtca
     6001 gactaaactg gctgacggaa tttatgcctc ttccgaccat caagcatttt atccgtactc
     6061 ctgatgatgc atggttactc accactgcga tccccggaaa aacagcattc caggtattag
     6121 aagaatatcc tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt
     6181 tgcattcgat tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt cgtctcgctc
     6241 aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag tgattttgat gacgagcgta
     6301 atggctggcc tgttgaacaa gtctggaaag aaatgcataa acttttgcca ttctcaccgg
     6361 attcagtcgt cactcatggt gatttctcac ttgataacct tatttttgac gaggggaaat
     6421 taataggttg tattgatgtt ggacgagtcg gaatcgcaga ccgataccag gatcttgcca
     6481 tcctatggaa ctgcctcggt gagttttctc cttcattaca gaaacggctt tttcaaaaat
     6541 atggtattga taatcctgat atgaataaat tgcagtttca tttgatgctc gatgagtttt
     6601 tctaatcaga attggttaat tggttgtaac actggcaaaa aggatctagg tgaagatcct
     6661 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     6721 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     6781 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     6841 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     6901 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     6961 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     7021 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     7081 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     7141 atgagaaagc gccacggttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     7201 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     7261 tcctgtcggg tttcgccacc tctg
//
