LOCUS       sequence-340509-        7650 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 7650)
  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 7650)
  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 -0
FEATURES             Location/Qualifiers
     source          1..7650
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     79..98
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     332..349
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    join(379..390,391..553)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     protein_bind    640..661
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(676..681,682..699,700..706)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    714..730
                     /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     719..741
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     738..754
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     738..754
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             750..1088
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWTCHTSGRITLNN
                     ANYLKLLS"
     promoter        775..793
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        complement(841..859)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(869..886)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(869..885)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(878..900)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     protein_bind    1048..1079
                     /label=rox
                     /bound_moiety="Dre recombinase"
     protein_bind    complement(1085..1118)
                     /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(1123..1156)
                     /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             1301..1357
                     /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             1358..2032
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="ASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAKL
                     KVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVV
                     TVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKMR
                     LKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHST
                     GA"
     CDS             1358..2032
                     /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(1485..1503)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1753..1772
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1947..1970
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     CDS             3907..3927
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3931..4632
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     protein_bind    5707..5740
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    5746..5779
                     /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    5786..5817
                     /label=rox
                     /bound_moiety="Dre recombinase"
     promoter        5983..6001
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(6024..6211,6212..6223)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(6277..6296)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             6288..7097
                     /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(6355..6374)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      join(7240..7650,1..178)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
       61 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
      121 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
      181 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
      241 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
      301 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaca
      361 gatttaaatg gtaccgagca gaggtgtaaa aagtactcaa aaattttact caagtgaaag
      421 tacaagtact tagggaaaat tttactcaat taaaagtaaa agtatctggc tagaatctta
      481 cttgagtaaa agtaaaaaag tactccatta aaattgtact tgagtattaa ggaagtaaaa
      541 gtaaaagcaa gaaagaaaac tagagtggtc tccctttagt gagggtcaat tgatgggagg
      601 cgttcgggcc acagcggaca cgcaggtgcg caacgcaatt aatgtgagtt agctcactca
      661 ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag
      721 cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagc gcgcaattaa
      781 ccctcactaa agggaacaaa agctggagga cctcgagggg gggcccggta cccaattcgc
      841 cctatagtga gtcgtattac gcgcgctcac tggccgtcgt tttacaacgt cgtgactggg
      901 aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc
      961 gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg
     1021 aatggacctg ccacacttcc ggccgcataa ctttaaataa tgccaattat ttaaagttac
     1081 tatcataact tcgtatagca tacattatac gaagttatca ccataacttc gtataggata
     1141 ctttatacga agttatccta ggcttaccac ccactagcgg tcagactgca gattgcagca
     1201 cgaaacagga agctgactcc acatggtcac atgctcactg aagtgttgac ttccctgaca
     1261 gctgtgcact ttctaaaccg gttttctcat tcatttacag gccacgaact tctctctgtt
     1321 aaagcaagca ggagacgtgg aagaaaaccc cggtcctgcc tcctccgagg acgtcatcaa
     1381 ggagttcatg cgcttcaagg tgcgcatgga gggctccgtg aacggccacg agttcgagat
     1441 cgagggcgag ggcgagggcc gcccctacga gggcacccag accgccaagc tgaaggtgac
     1501 caagggcggc cccctgccct tcgcctggga catcctgtcc cctcagttcc agtacggctc
     1561 caaggcctac gtgaagcacc ccgccgacat ccccgactac ttgaagctgt ccttccccga
     1621 gggcttcaag tgggagcgcg tgatgaactt cgaggacggc ggcgtggtga ccgtgaccca
     1681 ggactcctcc ctgcaggacg gcgagttcat ctacaaggtg aagctgcgcg gcaccaactt
     1741 cccctccgac ggccccgtaa tgcagaagaa gaccatgggc tgggaggcct ccaccgagcg
     1801 gatgtacccc gaggacggcg ccctgaaggg cgagatcaag atgaggctga agctgaagga
     1861 cggcggccac tacgacgccg aggtcaagac cacctacatg gccaagaagc ccgtgcagct
     1921 gcccggcgcc tacaagaccg acatcaagct ggacatcacc tcccacaacg aggactacac
     1981 catcgtggaa cagtacgagc gcgccgaggg ccgccactcc accggagcct aagactagta
     2041 attaagtctc agccaccgtt aactgaacat gtcaaaacct gtggagactg ttgagatttg
     2101 atgttctgaa aagataaagc ctataaataa aatgttgccc aaatttcctg cctgatgttt
     2161 ttctttgtct ttgctacatg gctttgctgc tcggatcggc tcactctgtg tatgccacgt
     2221 tcactttgta ctctccttct cacggtaggt ttattatttt tagatgtgca gttagtttct
     2281 gtgaaataac acaccacaca ctgatattgt ctgtgcattg acttggtgag tgcacattgt
     2341 ttttgatctt gacatattta tatttgattg atcaggtgaa ctgtgtgaat ctaaagtgct
     2401 ccatacagat gttctgcatt gaaaatattc tcattttatt agtggaagtg agtgtatgcc
     2461 acatccaatc aatttcagca aacaccccag tatgatttaa tgcaaaaaaa tgaaggtatc
     2521 aaacacgcat tactactttg cagttaaata tttaacattt attccaacac gaaaaaaagc
     2581 agtaaataac actttgacaa acacgtcagg acatcttatt tttgtcaccc tcacaggcaa
     2641 tttagtataa tatattatat atatatatat atcatataat aatattcagt ataatatata
     2701 tatatatatc atattataat attcagtata atataaaaca caaacacata tatgtataat
     2761 ataatataac atttttattt attgagatgc ctctatggac cgtgttataa gaagtaaaga
     2821 tcaggagaag taaacatgaa gtgtaattat gaatactgat gttaaattaa gctatgatga
     2881 gttttcactg ttaatttacc atctcaatta aatgttgatg cctccatgac caagttaagc
     2941 agatgagact gaggacaact gtagaagaca agatgttcac tttgctgaat atagctggct
     3001 tgacagttat ctatgactct ataaatatat atatattttt ttttttataa aatgatttat
     3061 ttataactat atatccattt ctcagacagg tgcttcatat ccctcactcc cgtagctgtc
     3121 catgctggat ctgtccccgt tgtttttaaa aagctaaata agttattaac atgactgcat
     3181 ccagcgagcc aaacctgtct ggtgtacagc taccagagaa gcttgagatc ctagtcaccg
     3241 gcgttcctag ttctttaaac tgttacaaag tgtttggacc agcaactgaa ttgcactgta
     3301 aattaccaca tcagggtctc cggggtcaac gctgctaagc aatggactgg aaaatttgtg
     3361 acattgagtt tttggaataa ctgggaatgc tggataactc tttgggtaaa gctatggaac
     3421 aaaagtacca gcatgaacta agaaatgact gtcacagcac aacatcaaac acaatgcaag
     3481 acaaaggttc tctgcttttg ccttacttga cagaaaaagt aagctctagg ttcatcgcat
     3541 gcagacagca gcaatacatg acatatataa aggctggctc ttttctccct tccattgaac
     3601 tgaaacttcc actcagtcag acttgcgggg gatccacagc ctgactaact cgacaaaccc
     3661 ccccaaacct aaggtgagtt gatctttaag ctttttacat tttcagctcg catatatcaa
     3721 ttcgcgcgca ttgtcacact agcacccact agcggtcaga ctgcagattg cagcacgaaa
     3781 caggaagctg actccacatg gtcacatgct cactgaagtg ttgacttccc tgacagctgt
     3841 gcactttcta aaccggtttt ctcattcatt tacagttcag ccggaattca cctcgagacc
     3901 atggctccaa agaagaagcg taaggtaacc atgagcgagc tgattaagga gaacatgcac
     3961 atgaagctgt acatggaggg caccgtggac aaccatcact tcaagtgcac atccgagggc
     4021 gaaggcaagc cctacgaggg cacccagacc atgagaatca aggtggtcga gggcggccct
     4081 ctccccttcg ccttcgacat cctggctact agcttcctct acggcagcaa gaccttcatc
     4141 aaccacaccc agggcatccc cgacttcttc aagcagtcct tccctgaggg cttcacatgg
     4201 gagagagtca ccacatacga agacgggggc gtgctgaccg ctacccagga caccagcctc
     4261 caggacggct gcctcatcta caacgtcaag atcagagggg tgaacttcac atccaacggc
     4321 cctgtgatgc agaagaaaac actcggctgg gaggccttca ccgagacgct gtaccccgct
     4381 gacggcggcc tggaaggcag aaacgacatg gccctgaagc tcgtgggcgg gagccatctg
     4441 atcgcaaaca tcaagaccac atatagatcc aagaaacccg ctaagaacct caagatgcct
     4501 ggcgtctact atgtggacta cagactggaa agaatcaagg aggccaacaa cgagacctac
     4561 gtcgagcagc acgaggtggc agtggccaga tactgcgacc tccctagcaa actggggcac
     4621 aagctcaatt aaagctgact aatttattta gcagtagata gctatattgt gtgaagtggt
     4681 aacagtccgt agatctacgg actgttacca cttcacgccg actcaaactg cgcagagaaa
     4741 cttcaaacga caacattggc atggcttttg ttatttttgg cgcttgactc aggatctaaa
     4801 aactggaacg gtgaaggtga cggcaatgtt ttttggcaaa taagcatccc cgaagttcta
     4861 caatgcatct gaggactcca tgttttgttt tctttagtca ttccaaatgt ttgttaaatg
     4921 cattgttccg aaacttattt gcctctatga aggctgccca gtaattggga gcatacttaa
     4981 cattgtagta ttgtatgtaa attatgtaac aaaacaatgt ctgggttttt gtacttttca
     5041 gccttaaaat cttgggttta tttttttttc tttgttccaa aaaaacgaag ctttaccatt
     5101 caagatgtaa aggtttccat tccccctggg catattgtaa aagctgtgtg gaacgtggcg
     5161 gtgccagaca tttggtgggg ccaacctgta cactgactaa ttcaattcca ataaaagtgc
     5221 acatgtaaga catcctaccc ctgtctcatt tcctgtcttg gctctgagtg gcttcaataa
     5281 aaatggcttt tgtgaagttg caccatgtta attacatttc agcactttca caccaggttt
     5341 ttgactaatt tgaatttatc gaagtttgac cttttttttt ttttttcttc aagctaattt
     5401 gaggttatca aaccatttca ttggaggaaa cttaatgttt aaaagcataa attcgaaaca
     5461 tgattcattt atttaactgc ttaatggtga aacaaacttc aagtaataca ccaagtgttc
     5521 cctcatatga aggtagctaa ctgcaaacgg ccttaacttt ccatcttaag ttgagctaca
     5581 taaaggcttt aacaggaagg aactgccagt ttaaaagtca aaccaccatg actgatataa
     5641 attcttgtcc atataacctt gcgtttcaca caatatagct atctactgct aaataaatta
     5701 gagctcataa cttcgtataa tgtatgctat acgaagttat gttagataac ttcgtataaa
     5761 gtatcctata cgaagttatg cgagctaact ttaaataatt ggcattattt aaagttatga
     5821 atcgataagt gaagagctat ccggatatat atgtatgtta tcataactgt actggctatg
     5881 tcaggtggta atgactgtaa cgttacgtta ctcgttgtag gcacgacatt gaatatgctc
     5941 ttctccgctg tggcccgaac gcctcccgct agcgatggga tctaatacga ctcactatag
     6001 ggagaccaag ggaaaataga atgaagtgat ctccaaaaaa taagtacttt ttgactgtaa
     6061 ataaaattgt aaggagtaaa aagtactttt ttttctaaaa aaatgtaatt aagtaaaagt
     6121 aaaagtattg atttttaatt gtactcaagt aaagtaaaaa tccccaaaaa taatacttaa
     6181 gtacagtaat caagtaaaat tactcaagta ctttacacct ctgggatcca gctgcatcat
     6241 gaacaataaa actgtctgct tacataaaca gtaatacaag gggtgttatg agccatattc
     6301 aacgggaaac gtcgaggccg cgattaaatt ccaacatgga tgctgattta tatgggtata
     6361 aatgggctcg cgataatgtc gggcaatcag gtgcgacaat ctatcgcttg tatgggaagc
     6421 ccgatgcgcc agagttgttt ctgaaacatg gcaaaggtag cgttgccaat gatgttacag
     6481 atgagatggt cagactaaac tggctgacgg aatttatgcc tcttccgacc atcaagcatt
     6541 ttatccgtac tcctgatgat gcatggttac tcaccactgc gatccccgga aaaacagcat
     6601 tccaggtatt agaagaatat cctgattcag gtgaaaatat tgttgatgcg ctggcagtgt
     6661 tcctgcgccg gttgcattcg attcctgttt gtaattgtcc ttttaacagc gatcgcgtat
     6721 ttcgtctcgc tcaggcgcaa tcacgaatga ataacggttt ggttgatgcg agtgattttg
     6781 atgacgagcg taatggctgg cctgttgaac aagtctggaa agaaatgcat aaacttttgc
     6841 cattctcacc ggattcagtc gtcactcatg gtgatttctc acttgataac cttatttttg
     6901 acgaggggaa attaataggt tgtattgatg ttggacgagt cggaatcgca gaccgatacc
     6961 aggatcttgc catcctatgg aactgcctcg gtgagttttc tccttcatta cagaaacggc
     7021 tttttcaaaa atatggtatt gataatcctg atatgaataa attgcagttt catttgatgc
     7081 tcgatgagtt tttctaatca gaattggtta attggttgta acactggcaa aaaggatcta
     7141 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     7201 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     7261 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     7321 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     7381 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     7441 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     7501 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     7561 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     7621 acagcgtgag ctatgagaaa gcgccacggt
//
