LOCUS       sequence-353847-        7375 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 7375)
  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 7375)
  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-mRFP-; HE1A:BFP -2
FEATURES             Location/Qualifiers
     source          1..7375
                     /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..980
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWTCHTSGRITLNN
                     ANYLKLLS"
     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    940..971
                     /label=rox
                     /bound_moiety="Dre recombinase"
     protein_bind    complement(977..1010)
                     /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(1015..1048)
                     /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..1925
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="ASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAKL
                     KVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVV
                     TVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKMR
                     LKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHST
                     GA"
     CDS             1251..1925
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /note="mammalian codon-optimized"
                     /translation="ASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAKL
                     KVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVV
                     TVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKMR
                     LKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHST
                     GA"
     primer_bind     complement(1378..1396)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1646..1665
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1840..1863
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     CDS             3525..3545
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3549..4250
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     protein_bind    5324..5357
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    5363..5396
                     /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    5403..5434
                     /label=rox
                     /bound_moiety="Dre recombinase"
     promoter        5600..5618
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(5641..5828,5829..5840)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(5894..5913)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5905..6714
                     /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(5972..5991)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      join(6857..7375,1..70)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7346..7365
                     /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 cgaatggacc tgccacactt ccggccgcat aactttaaat aatgccaatt
      961 atttaaagtt actatcataa cttcgtatag catacattat acgaagttat caccataact
     1021 tcgtatagga tactttatac gaagttatcc taggcttacc acccactagc ggtcagactg
     1081 cagattgcag cacgaaacag gaagctgact ccacatggtc acatgctcac tgaagtgttg
     1141 acttccctga cagctgtgca ctttctaaac cggttttctc attcatttac agcgccacga
     1201 acttctctct gttaaagcaa gcaggagacg tggaagaaaa ccccggtcct gcctcctccg
     1261 aggacgtcat caaggagttc atgcgcttca aggtgcgcat ggagggctcc gtgaacggcc
     1321 acgagttcga gatcgagggc gagggcgagg gccgccccta cgagggcacc cagaccgcca
     1381 agctgaaggt gaccaagggc ggccccctgc ccttcgcctg ggacatcctg tcccctcagt
     1441 tccagtacgg ctccaaggcc tacgtgaagc accccgccga catccccgac tacttgaagc
     1501 tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac ggcggcgtgg
     1561 tgaccgtgac ccaggactcc tccctgcagg acggcgagtt catctacaag gtgaagctgc
     1621 gcggcaccaa cttcccctcc gacggccccg taatgcagaa gaagaccatg ggctgggagg
     1681 cctccaccga gcggatgtac cccgaggacg gcgccctgaa gggcgagatc aagatgaggc
     1741 tgaagctgaa ggacggcggc cactacgacg ccgaggtcaa gaccacctac atggccaaga
     1801 agcccgtgca gctgcccggc gcctacaaga ccgacatcaa gctggacatc acctcccaca
     1861 acgaggacta caccatcgtg gaacagtacg agcgcgccga gggccgccac tccaccggag
     1921 cctaagacta gtaattaagt ctcagccacc gttaactgaa catgtcaaaa cctgtggaga
     1981 ctgttgagat ttgatgttct gaaaagataa agcctataaa taaaatgttg cccaaatttc
     2041 ctgcctgatg tttttctttg tctttgctac atggctttgc tgctcggatc ggctcactct
     2101 gtgtatgcca cgttcacttt gtactctcct tctcacggta ggtttattat ttttagatgt
     2161 gcagttagtt tctgtgaaat aacacaccac acactgatat tgtctgtgca ttgacttggt
     2221 gagtgcacat tgtttttgat cttgacatat ttatatttga ttgatcaggt gaactgtgtg
     2281 aatctaaagt gctccataca gatgttctgc attgaaaata ttctcatttt attagtggaa
     2341 gtgagtgtat gccacatcca atcaatttca gcaaacaccc cagtatgatt taatgcaaaa
     2401 aaatgaaggt atcaaacacg cattactact ttgcagttaa atatttaaca tttattccaa
     2461 cacgaaaaaa agcagtaaat aacactttga caaacacgtc aggacatctt atttttgtca
     2521 ccctcacagg caatttagta taatatatta tatatatata tatatcatat aataatattc
     2581 agtataatat atatatatat atcatattat aatattcagt ataatataaa acacaaacac
     2641 atatatgtat aatataatat aacattttta tttattgaga tgcctctatg gaccgtgtta
     2701 taagaagtaa agatcaggag aagtaaacat gaagtgtaat tatgaatact gatgttaaat
     2761 taagctatga tgagttttca ctgttaattt accatctcaa ttaaatgttg atgcctccat
     2821 gaccaagtta agcagatgag actgaggaca actgtagaag acaagatgtt cactttgctg
     2881 aatatagctg gcttgacagt tatctatgac tctataaata tatatatatt ttttttttta
     2941 taaaatgatt tatttataac tatatatcca tttctcagac aggtgcttca tatccctcac
     3001 tcccgtagct gtccatgctg gatctgtccc cgttgttttt aaaaagctaa ataagttatt
     3061 aacatgactg catccagcga gccaaacctg tctggtgtac agctaccaga gaagcttgag
     3121 atcctagtca aagctttcaa ccactccagg catagctaga gccactgatg catctataga
     3181 gctgaagtaa agatagggtt gatgcaaatc gatttagaat tttcaatgta gaaaaaacaa
     3241 aataaaaagt ctattcactt gatggataga ttagtcaaac aaacatagag taggcacttc
     3301 ccttgactct gtaaactttt ggatttaaca gattgtgcac tccttttagt taaacacatt
     3361 agtccctgaa aactaagacc ctaaccataa tcccattgta gacccaccta ctcgggtgaa
     3421 tataaagctc gcacagatca tctcacagac atcagacctt ggcaaaggag catcctgaac
     3481 atctctacac actgaggcaa cgaattcacc tcgagaccat ggctccaaag aagaagcgta
     3541 aggtaaccat gagcgagctg attaaggaga acatgcacat gaagctgtac atggagggca
     3601 ccgtggacaa ccatcacttc aagtgcacat ccgagggcga aggcaagccc tacgagggca
     3661 cccagaccat gagaatcaag gtggtcgagg gcggccctct ccccttcgcc ttcgacatcc
     3721 tggctactag cttcctctac ggcagcaaga ccttcatcaa ccacacccag ggcatccccg
     3781 acttcttcaa gcagtccttc cctgagggct tcacatggga gagagtcacc acatacgaag
     3841 acgggggcgt gctgaccgct acccaggaca ccagcctcca ggacggctgc ctcatctaca
     3901 acgtcaagat cagaggggtg aacttcacat ccaacggccc tgtgatgcag aagaaaacac
     3961 tcggctggga ggccttcacc gagacgctgt accccgctga cggcggcctg gaaggcagaa
     4021 acgacatggc cctgaagctc gtgggcggga gccatctgat cgcaaacatc aagaccacat
     4081 atagatccaa gaaacccgct aagaacctca agatgcctgg cgtctactat gtggactaca
     4141 gactggaaag aatcaaggag gccaacaacg agacctacgt cgagcagcac gaggtggcag
     4201 tggccagata ctgcgacctc cctagcaaac tggggcacaa gctcaattaa agctgactaa
     4261 tttatttagc agtagatagc tatattgtgt gaagtggtaa cagtccgtag atctacggac
     4321 tgttaccact tcacgccgac tcaaactgcg cagagaaact tcaaacgaca acattggcat
     4381 ggcttttgtt atttttggcg cttgactcag gatctaaaaa ctggaacggt gaaggtgacg
     4441 gcaatgtttt ttggcaaata agcatccccg aagttctaca atgcatctga ggactccatg
     4501 ttttgttttc tttagtcatt ccaaatgttt gttaaatgca ttgttccgaa acttatttgc
     4561 ctctatgaag gctgcccagt aattgggagc atacttaaca ttgtagtatt gtatgtaaat
     4621 tatgtaacaa aacaatgtct gggtttttgt acttttcagc cttaaaatct tgggtttatt
     4681 tttttttctt tgttccaaaa aaacgaagct ttaccattca agatgtaaag gtttccattc
     4741 cccctgggca tattgtaaaa gctgtgtgga acgtggcggt gccagacatt tggtggggcc
     4801 aacctgtaca ctgactaatt caattccaat aaaagtgcac atgtaagaca tcctacccct
     4861 gtctcatttc ctgtcttggc tctgagtggc ttcaataaaa atggcttttg tgaagttgca
     4921 ccatgttaat tacatttcag cactttcaca ccaggttttt gactaatttg aatttatcga
     4981 agtttgacct tttttttttt tttcttcaag ctaatttgag gttatcaaac catttcattg
     5041 gaggaaactt aatgtttaaa agcataaatt cgaaacatga ttcatttatt taactgctta
     5101 atggtgaaac aaacttcaag taatacacca agtgttccct catatgaagg tagctaactg
     5161 caaacggcct taactttcca tcttaagttg agctacataa aggctttaac aggaaggaac
     5221 tgccagttta aaagtcaaac caccatgact gatataaatt cttgtccata taaccttgcg
     5281 tttcacacaa tatagctatc tactgctaaa taaattagag ctcataactt cgtataatgt
     5341 atgctatacg aagttatgtt agataacttc gtataaagta tcctatacga agttatgcga
     5401 gctaacttta aataattggc attatttaaa gttatgaatc gataagtgaa gagctatccg
     5461 gatatatatg tatgttatca taactgtact ggctatgtca ggtggtaatg actgtaacgt
     5521 tacgttactc gttgtaggca cgacattgaa tatgctcttc tccgctgtgg cccgaacgcc
     5581 tcccgctagc gatgggatct aatacgactc actataggga gaccaaggga aaatagaatg
     5641 aagtgatctc caaaaaataa gtactttttg actgtaaata aaattgtaag gagtaaaaag
     5701 tacttttttt tctaaaaaaa tgtaattaag taaaagtaaa agtattgatt tttaattgta
     5761 ctcaagtaaa gtaaaaatcc ccaaaaataa tacttaagta cagtaatcaa gtaaaattac
     5821 tcaagtactt tacacctctg ggatccagct gcatcatgaa caataaaact gtctgcttac
     5881 ataaacagta atacaagggg tgttatgagc catattcaac gggaaacgtc gaggccgcga
     5941 ttaaattcca acatggatgc tgatttatat gggtataaat gggctcgcga taatgtcggg
     6001 caatcaggtg cgacaatcta tcgcttgtat gggaagcccg atgcgccaga gttgtttctg
     6061 aaacatggca aaggtagcgt tgccaatgat gttacagatg agatggtcag actaaactgg
     6121 ctgacggaat ttatgcctct tccgaccatc aagcatttta tccgtactcc tgatgatgca
     6181 tggttactca ccactgcgat ccccggaaaa acagcattcc aggtattaga agaatatcct
     6241 gattcaggtg aaaatattgt tgatgcgctg gcagtgttcc tgcgccggtt gcattcgatt
     6301 cctgtttgta attgtccttt taacagcgat cgcgtatttc gtctcgctca ggcgcaatca
     6361 cgaatgaata acggtttggt tgatgcgagt gattttgatg acgagcgtaa tggctggcct
     6421 gttgaacaag tctggaaaga aatgcataaa cttttgccat tctcaccgga ttcagtcgtc
     6481 actcatggtg atttctcact tgataacctt atttttgacg aggggaaatt aataggttgt
     6541 attgatgttg gacgagtcgg aatcgcagac cgataccagg atcttgccat cctatggaac
     6601 tgcctcggtg agttttctcc ttcattacag aaacggcttt ttcaaaaata tggtattgat
     6661 aatcctgata tgaataaatt gcagtttcat ttgatgctcg atgagttttt ctaatcagaa
     6721 ttggttaatt ggttgtaaca ctggcaaaaa ggatctaggt gaagatcctt tttgataatc
     6781 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     6841 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     6901 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     6961 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
     7021 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     7081 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     7141 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     7201 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     7261 ccacggttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     7321 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgt
//
