LOCUS Exported 5222 bp ds-DNA circular SYN 01-MAR-2022 DEFINITION Expresses zebrafish ythdf1 with 3x flag tag, for mRNA injection. ACCESSION . VERSION . KEYWORDS pSP64T-ythdf1-3xflag SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5222) AUTHORS Kontur C, Jeong M, Cifuentes D, Giraldez AJ TITLE Ythdf m(6)A Readers Function Redundantly during Zebrafish Development. JOURNAL Cell Rep. 2020 Dec 29;33(13):108598. doi: 10.1016/j.celrep.2020.108598. PUBMED 33378672 REFERENCE 2 (bases 1 to 5222) AUTHORS . TITLE Direct Submission JOURNAL Exported Mar 1, 2022 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5222 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(11..28) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(182..770) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(262..281) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(941..1801) /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind 1564..1583 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(1802..1906) /gene="bla" /label=AmpR promoter primer_bind 1974..1992 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind complement(2030..2052) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 2152..2171 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 2385..2404 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" promoter 2694..2712 /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind 2694..2711 /label=SP6 /note="SP6 promoter, forward primer" 5'UTR 2724..2766 /label=Xenopus globin 5'-UTR /note="translational enhancer from the 5'-UTR of the major beta-globin gene of Xenopus laevis" regulatory 2777..2786 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 2786..2851 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" 3'UTR 4749..4874 /label=Xenopus globin 3'-UTR /note="3'-UTR of the major beta-globin gene of Xenopus laevis" primer_bind complement(5002..5018) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5002..5018) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(5015..5037) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 5026..5042 /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)." promoter complement(5050..5080) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5095..5116 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." ORIGIN 1 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat 61 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga 121 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt 181 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt 241 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc 301 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa 361 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct 421 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta 481 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg 541 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc 601 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta 661 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg 721 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt 781 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg 841 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta 901 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg 961 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg 1021 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc 1081 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg 1141 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg 1201 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag 1261 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat 1321 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc 1381 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc 1441 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa 1501 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac 1561 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt 1621 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc 1681 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa 1741 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca 1801 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat 1861 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa 1921 aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc 1981 gtatcacgag gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca 2041 tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc 2101 gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg gcttaactat gcggcatcag 2161 agcagattgt actgagagtg caccattcga cgctctccct tatgcgactc ctgcattagg 2221 aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc 2281 aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa 2341 caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata 2401 taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag 2461 aggatctggc tagcgatgac cctgctgatt ggttcgctga ccatttccgg gtgcgggacg 2521 gcgttaccag aaactcagaa ggttcgtcca accaaaccga ctctgacggc agtttacgag 2581 agagatgata gggtctgctt cagtaagcca gatgctacac aattaggctt gtacatattg 2641 tcgttagaac gcggctacaa ttaatacata accttatgta tcatacacat acgatttagg 2701 tgacactata gaatacaagc ttgcttgttc tttttgcaga agctcagaat aaacgctcaa 2761 ctttggcaga tccgcggccg ccatggacta caaagaccat gacggtgatt ataaagatca 2821 tgacatcgac tacaaggatg acgatgacaa gaattcaggt tctggctcag gatcgtctac 2881 caccagtatt gacccacaga catcaaaagg gcaagacgct aaagtgcaaa atggctctct 2941 tcatcagaag gaaacggtcc acgacaatga ctttgaacca tacctcactg gccagtctaa 3001 tccgaataac agttaccaat ccatgaccga cccatacctg tctagctact acgccccctc 3061 aattgggttc ccgtaccccc tcagcgaggc tccctggtcc acaggtggag acccgccgat 3121 cccatacctc actccgtatg cccccctcag caatggagac catcacttca tgcatgacac 3181 tgtgtttgga cagccggggg gtctgggcag tagcatttat ccacaccgct ttaacttttt 3241 ccccgaaaac cctgcgtttt ctgcctgggg tacgagtggc tcccagggcc agcagactca 3301 gagttctgca tatgggggca gctacagtta cccgcccagt tctttaggtg gaaccctggt 3361 gccagacggg caaacaggct tccctagtga caccctgaat aaggcaccag gtatgaatag 3421 cttggagcag ggcatggtgg ggctgaagat cggtggtgat gtgactggag gtggttccgg 3481 agttaagacg gtgggctccg tgattggagg agggccggtg gctgcggcgg tggccacggg 3541 taatggtggg actcctattg gactgccgcc ccctaagccc acatcttggg cagccattgc 3601 tagcaaaccg gccaagcctc agcaactgaa ggtgaagagt aagccaggga tgcccatggc 3661 tggaggcacc ctgcctccgc cgcccatcaa acacaacatg gacattggca cttgggataa 3721 taaggggcct gtgaccaaag tggcatcatc attgccacct caccaccacc accagcagca 3781 gcctcccctc cactcccagg gtcaccttcc acctcaccct catggactgc cacccccacc 3841 tcagccccca atgccgtctg cacaatcact tgcccagcag atggccatgc agggcccacc 3901 ccctccgcag ccttaccaga accacgcccc tgctcctccc ccgcaaactc ggtgggttgc 3961 cccacgcaat cgcaaccctg gttatggtgg tggaggaagc gtggacagca gtggctcctc 4021 gagtggtgga ggggtcggca atggaggcgg aggtggccca ccgggctccg gcgccatcga 4081 gtcccatccc gtcttagaaa agctgcgtgc ggcacacagc tacaacccca aggagttcga 4141 ttggaacctg aaaaatggcc gggtgttcat cattaagagc tactctgagg acgacatcca 4201 tcgctccatc aagtactcca tctggtgcag cacggaacat ggaaacaagc ggctggattc 4261 tgcgtttcgt gccatcaacg gcaaaggtcc tgtctacctg ctgttcagcg tcaacggcag 4321 cggacacttc tgtggcgtgg cggagatgcg ttcgcctgtt gactacggca ccagtgccgg 4381 tgtttgggcg caggacaagt ggaagggcaa atttgatgtg gactggctgt ttgttaaaga 4441 cgtgcccaac agtcagctca ggcacatccg cctggagaac aatgacaaca agcccgtgac 4501 caactcgcgt gacacacagg aggtgcctct ggaaaaggcc aagcaggtgc tgaagatcat 4561 cgctacctac aaacacacaa cctccatctt cgatgacttc tcacattacg agaaacggca 4621 ggaggaggaa gaggtggtaa gaaagaatct tgagcctgct cctatccaga accgatcccg 4681 actggatcaa gaacgccaga accgaaataa acaatagctc gagaataatt cggatctggt 4741 taccactaaa ccagcctcaa gaacacccga atggagtctc taagctacat aataccaact 4801 tacactttac aaaatgttgt cccccaaaat gtagccattc gtatctgctc ctaataaaaa 4861 gaaagtttct tcacattcta aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4921 aaaaaaaaaa mccccccccc cccccccctg caggtcgact ctagaggatc cccgggcgag 4981 ctcgaattaa ttcgtaatca tgtcatagct gtttcctgtg tgaaattgtt atccgctcac 5041 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt 5101 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc 5161 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg 5221 ct //