LOCUS Exported 7284 bp ds-DNA circular SYN 07-FEB-2022 DEFINITION Donor for generating conditional alleles in zebrafish using precise CRISPR directed genomic integration with the GeneWeld method. ACCESSION . VERSION . KEYWORDS pUFlip-floxed2A-KalTA4-; HE1A:BFP -2 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7284) AUTHORS Almeida MP, Kambakam SE, Liu F, Ming Z, Welker JM, Wierson WA, Schultz-Rogers LE, Ekker SC, Clark KJ, Essner JJ, McGrail MP TITLE Zebrafish Cre/lox regulated UFlip alleles generated by CRISPR/Cas targeted integration provide cell-type specific conditional gene inactivation JOURNAL bioRxiv REFERENCE 2 (bases 1 to 7284) AUTHORS . TITLE Direct Submission JOURNAL Exported Feb 7, 2022 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7284 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 206..223 /label=L4440 /note="L4440 vector, forward primer" 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 550..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" primer_bind 647..667 /label=T3 /note="T3 promoter, forward primer" 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 960..993 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." protein_bind 998..1031 /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 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 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 complement(5215..5248) /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." protein_bind complement(5254..5287) /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 5491..5509 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" 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 6748..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 //