LOCUS       sequence-340510-        7652 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 7652)
  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 7652)
  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-; gcry1:BFP -1
FEATURES             Location/Qualifiers
     source          1..7652
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     161..178
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    join(208..219,220..382)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     protein_bind    469..490
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(505..510,511..528,529..535)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    543..559
                     /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     548..570
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     567..583
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     567..583
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             579..917
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWTCHTSGRITLNN
                     ANYLKLLS"
     promoter        604..622
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        complement(670..688)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(698..715)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(698..714)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(707..729)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     protein_bind    877..908
                     /label=rox
                     /bound_moiety="Dre recombinase"
     protein_bind    complement(914..947)
                     /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(952..985)
                     /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             1132..1188
                     /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             1189..1863
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="ASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAKL
                     KVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVV
                     TVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKMR
                     LKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHST
                     GA"
     CDS             1189..1863
                     /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(1316..1334)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1584..1603
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1778..1801
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     CDS             3738..3758
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3762..4463
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     protein_bind    5538..5571
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    5577..5610
                     /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    5617..5648
                     /label=rox
                     /bound_moiety="Dre recombinase"
     promoter        5814..5832
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(5855..6042,6043..6054)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(6108..6127)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             6119..6928
                     /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(6186..6205)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      join(7071..7652,1..7)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7560..7579
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
       61 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
      121 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
      181 agcggaacag atttaaatgg taccgagcag aggtgtaaaa agtactcaaa aattttactc
      241 aagtgaaagt acaagtactt agggaaaatt ttactcaatt aaaagtaaaa gtatctggct
      301 agaatcttac ttgagtaaaa gtaaaaaagt actccattaa aattgtactt gagtattaag
      361 gaagtaaaag taaaagcaag aaagaaaact agagtggtct ccctttagtg agggtcaatt
      421 gatgggaggc gttcgggcca cagcggacac gcaggtgcgc aacgcaatta atgtgagtta
      481 gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg
      541 aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagcg
      601 cgcaattaac cctcactaaa gggaacaaaa gctggaggac ctcgaggggg ggcccggtac
      661 ccaattcgcc ctatagtgag tcgtattacg cgcgctcact ggccgtcgtt ttacaacgtc
      721 gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg
      781 ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc
      841 tgaatggcga atggacctgc cacacttccg gccgcataac tttaaataat gccaattatt
      901 taaagttact atcataactt cgtatagcat acattatacg aagttatcac cataacttcg
      961 tataggatac tttatacgaa gttatcctag gcttaccacc cactagcggt cagactgcag
     1021 attgcagcac gaaacaggaa gctgactcca catggtcaca tgctcactga agtgttgact
     1081 tccctgacag ctgtgcactt tctaaaccgg ttttctcatt catttacagt tgccacgaac
     1141 ttctctctgt taaagcaagc aggagacgtg gaagaaaacc ccggtcctgc ctcctccgag
     1201 gacgtcatca aggagttcat gcgcttcaag gtgcgcatgg agggctccgt gaacggccac
     1261 gagttcgaga tcgagggcga gggcgagggc cgcccctacg agggcaccca gaccgccaag
     1321 ctgaaggtga ccaagggcgg ccccctgccc ttcgcctggg acatcctgtc ccctcagttc
     1381 cagtacggct ccaaggccta cgtgaagcac cccgccgaca tccccgacta cttgaagctg
     1441 tccttccccg agggcttcaa gtgggagcgc gtgatgaact tcgaggacgg cggcgtggtg
     1501 accgtgaccc aggactcctc cctgcaggac ggcgagttca tctacaaggt gaagctgcgc
     1561 ggcaccaact tcccctccga cggccccgta atgcagaaga agaccatggg ctgggaggcc
     1621 tccaccgagc ggatgtaccc cgaggacggc gccctgaagg gcgagatcaa gatgaggctg
     1681 aagctgaagg acggcggcca ctacgacgcc gaggtcaaga ccacctacat ggccaagaag
     1741 cccgtgcagc tgcccggcgc ctacaagacc gacatcaagc tggacatcac ctcccacaac
     1801 gaggactaca ccatcgtgga acagtacgag cgcgccgagg gccgccactc caccggagcc
     1861 taagactagt aattaagtct cagccaccgt taactgaaca tgtcaaaacc tgtggagact
     1921 gttgagattt gatgttctga aaagataaag cctataaata aaatgttgcc caaatttcct
     1981 gcctgatgtt tttctttgtc tttgctacat ggctttgctg ctcggatcgg ctcactctgt
     2041 gtatgccacg ttcactttgt actctccttc tcacggtagg tttattattt ttagatgtgc
     2101 agttagtttc tgtgaaataa cacaccacac actgatattg tctgtgcatt gacttggtga
     2161 gtgcacattg tttttgatct tgacatattt atatttgatt gatcaggtga actgtgtgaa
     2221 tctaaagtgc tccatacaga tgttctgcat tgaaaatatt ctcattttat tagtggaagt
     2281 gagtgtatgc cacatccaat caatttcagc aaacacccca gtatgattta atgcaaaaaa
     2341 atgaaggtat caaacacgca ttactacttt gcagttaaat atttaacatt tattccaaca
     2401 cgaaaaaaag cagtaaataa cactttgaca aacacgtcag gacatcttat ttttgtcacc
     2461 ctcacaggca atttagtata atatattata tatatatata tatcatataa taatattcag
     2521 tataatatat atatatatat catattataa tattcagtat aatataaaac acaaacacat
     2581 atatgtataa tataatataa catttttatt tattgagatg cctctatgga ccgtgttata
     2641 agaagtaaag atcaggagaa gtaaacatga agtgtaatta tgaatactga tgttaaatta
     2701 agctatgatg agttttcact gttaatttac catctcaatt aaatgttgat gcctccatga
     2761 ccaagttaag cagatgagac tgaggacaac tgtagaagac aagatgttca ctttgctgaa
     2821 tatagctggc ttgacagtta tctatgactc tataaatata tatatatttt tttttttata
     2881 aaatgattta tttataacta tatatccatt tctcagacag gtgcttcata tccctcactc
     2941 ccgtagctgt ccatgctgga tctgtccccg ttgtttttaa aaagctaaat aagttattaa
     3001 catgactgca tccagcgagc caaacctgtc tggtgtacag ctaccagaga agcttgagat
     3061 cctagtcacc ggcgttccta gttctttaaa ctgttacaaa gtgtttggac cagcaactga
     3121 attgcactgt aaattaccac atcagggtct ccggggtcaa cgctgctaag caatggactg
     3181 gaaaatttgt gacattgagt ttttggaata actgggaatg ctggataact ctttgggtaa
     3241 agctatggaa caaaagtacc agcatgaact aagaaatgac tgtcacagca caacatcaaa
     3301 cacaatgcaa gacaaaggtt ctctgctttt gccttacttg acagaaaaag taagctctag
     3361 gttcatcgca tgcagacagc agcaatacat gacatatata aaggctggct cttttctccc
     3421 ttccattgaa ctgaaacttc cactcagtca gacttgcggg ggatccacag cctgactaac
     3481 tcgacaaacc cccccaaacc taaggtgagt tgatctttaa gctttttaca ttttcagctc
     3541 gcatatatca attcgcgcgc attgtcacac tagcacccac tagcggtcag actgcagatt
     3601 gcagcacgaa acaggaagct gactccacat ggtcacatgc tcactgaagt gttgacttcc
     3661 ctgacagctg tgcactttct aaaccggttt tctcattcat ttacagttca gccggaattc
     3721 acctcgagac catggctcca aagaagaagc gtaaggtaac catgagcgag ctgattaagg
     3781 agaacatgca catgaagctg tacatggagg gcaccgtgga caaccatcac ttcaagtgca
     3841 catccgaggg cgaaggcaag ccctacgagg gcacccagac catgagaatc aaggtggtcg
     3901 agggcggccc tctccccttc gccttcgaca tcctggctac tagcttcctc tacggcagca
     3961 agaccttcat caaccacacc cagggcatcc ccgacttctt caagcagtcc ttccctgagg
     4021 gcttcacatg ggagagagtc accacatacg aagacggggg cgtgctgacc gctacccagg
     4081 acaccagcct ccaggacggc tgcctcatct acaacgtcaa gatcagaggg gtgaacttca
     4141 catccaacgg ccctgtgatg cagaagaaaa cactcggctg ggaggccttc accgagacgc
     4201 tgtaccccgc tgacggcggc ctggaaggca gaaacgacat ggccctgaag ctcgtgggcg
     4261 ggagccatct gatcgcaaac atcaagacca catatagatc caagaaaccc gctaagaacc
     4321 tcaagatgcc tggcgtctac tatgtggact acagactgga aagaatcaag gaggccaaca
     4381 acgagaccta cgtcgagcag cacgaggtgg cagtggccag atactgcgac ctccctagca
     4441 aactggggca caagctcaat taaagctgac taatttattt agcagtagat agctatattg
     4501 tgtgaagtgg taacagtccg tagatctacg gactgttacc acttcacgcc gactcaaact
     4561 gcgcagagaa acttcaaacg acaacattgg catggctttt gttatttttg gcgcttgact
     4621 caggatctaa aaactggaac ggtgaaggtg acggcaatgt tttttggcaa ataagcatcc
     4681 ccgaagttct acaatgcatc tgaggactcc atgttttgtt ttctttagtc attccaaatg
     4741 tttgttaaat gcattgttcc gaaacttatt tgcctctatg aaggctgccc agtaattggg
     4801 agcatactta acattgtagt attgtatgta aattatgtaa caaaacaatg tctgggtttt
     4861 tgtacttttc agccttaaaa tcttgggttt attttttttt ctttgttcca aaaaaacgaa
     4921 gctttaccat tcaagatgta aaggtttcca ttccccctgg gcatattgta aaagctgtgt
     4981 ggaacgtggc ggtgccagac atttggtggg gccaacctgt acactgacta attcaattcc
     5041 aataaaagtg cacatgtaag acatcctacc cctgtctcat ttcctgtctt ggctctgagt
     5101 ggcttcaata aaaatggctt ttgtgaagtt gcaccatgtt aattacattt cagcactttc
     5161 acaccaggtt tttgactaat ttgaatttat cgaagtttga cctttttttt tttttttctt
     5221 caagctaatt tgaggttatc aaaccatttc attggaggaa acttaatgtt taaaagcata
     5281 aattcgaaac atgattcatt tatttaactg cttaatggtg aaacaaactt caagtaatac
     5341 accaagtgtt ccctcatatg aaggtagcta actgcaaacg gccttaactt tccatcttaa
     5401 gttgagctac ataaaggctt taacaggaag gaactgccag tttaaaagtc aaaccaccat
     5461 gactgatata aattcttgtc catataacct tgcgtttcac acaatatagc tatctactgc
     5521 taaataaatt agagctcata acttcgtata atgtatgcta tacgaagtta tgttagataa
     5581 cttcgtataa agtatcctat acgaagttat gcgagctaac tttaaataat tggcattatt
     5641 taaagttatg aatcgataag tgaagagcta tccggatata tatgtatgtt atcataactg
     5701 tactggctat gtcaggtggt aatgactgta acgttacgtt actcgttgta ggcacgacat
     5761 tgaatatgct cttctccgct gtggcccgaa cgcctcccgc tagcgatggg atctaatacg
     5821 actcactata gggagaccaa gggaaaatag aatgaagtga tctccaaaaa ataagtactt
     5881 tttgactgta aataaaattg taaggagtaa aaagtacttt tttttctaaa aaaatgtaat
     5941 taagtaaaag taaaagtatt gatttttaat tgtactcaag taaagtaaaa atccccaaaa
     6001 ataatactta agtacagtaa tcaagtaaaa ttactcaagt actttacacc tctgggatcc
     6061 agctgcatca tgaacaataa aactgtctgc ttacataaac agtaatacaa ggggtgttat
     6121 gagccatatt caacgggaaa cgtcgaggcc gcgattaaat tccaacatgg atgctgattt
     6181 atatgggtat aaatgggctc gcgataatgt cgggcaatca ggtgcgacaa tctatcgctt
     6241 gtatgggaag cccgatgcgc cagagttgtt tctgaaacat ggcaaaggta gcgttgccaa
     6301 tgatgttaca gatgagatgg tcagactaaa ctggctgacg gaatttatgc ctcttccgac
     6361 catcaagcat tttatccgta ctcctgatga tgcatggtta ctcaccactg cgatccccgg
     6421 aaaaacagca ttccaggtat tagaagaata tcctgattca ggtgaaaata ttgttgatgc
     6481 gctggcagtg ttcctgcgcc ggttgcattc gattcctgtt tgtaattgtc cttttaacag
     6541 cgatcgcgta tttcgtctcg ctcaggcgca atcacgaatg aataacggtt tggttgatgc
     6601 gagtgatttt gatgacgagc gtaatggctg gcctgttgaa caagtctgga aagaaatgca
     6661 taaacttttg ccattctcac cggattcagt cgtcactcat ggtgatttct cacttgataa
     6721 ccttattttt gacgagggga aattaatagg ttgtattgat gttggacgag tcggaatcgc
     6781 agaccgatac caggatcttg ccatcctatg gaactgcctc ggtgagtttt ctccttcatt
     6841 acagaaacgg ctttttcaaa aatatggtat tgataatcct gatatgaata aattgcagtt
     6901 tcatttgatg ctcgatgagt ttttctaatc agaattggtt aattggttgt aacactggca
     6961 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     7021 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     7081 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     7141 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     7201 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     7261 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     7321 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     7381 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     7441 ctgagatacc tacagcgtga gctatgagaa agcgccacgg ttcccgaagg gagaaaggcg
     7501 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     7561 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     7621 tttttgtgat gctcgtcagg ggggcggagc ct
//
