LOCUS Exported 7650 bp ds-DNA circular SYN 20-AUG-2021 DEFINITION Donor for generating conditional alleles in zebrafish using precise CRISPR directed genomic integration with the GeneWeld method. ACCESSION . VERSION . KEYWORDS pUFlip-floxed2A-mRFP-; gcry1:BFP -0 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7650) TITLE Zebrafish Cre/lox regulated UFlip alleles generated by CRISPR/Cas targeted integration provide cell-type specific conditional gene inactivation JOURNAL Unpublished REFERENCE 2 (bases 1 to 7650) AUTHORS . TITLE Direct Submission JOURNAL Exported Aug 20, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7650 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 79..98 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 332..349 /label=L4440 /note="L4440 vector, forward primer" 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 676..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" primer_bind 773..793 /label=T3 /note="T3 promoter, forward primer" 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 1085..1118 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." protein_bind 1123..1156 /label=lox2272 /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GGATACTT). 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 /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 complement(5707..5740) /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." protein_bind complement(5746..5779) /label=lox2272 /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GGATACTT). lox2272 sites are compatible with each other, but incompatible with loxP or loxN sites (Lee and Saito, 1988)." promoter 5983..6001 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" 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 7240..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 //