LOCUS Exported 6201 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expression of sgRNA precursor targeted against ade6+. ACCESSION . VERSION . KEYWORDS pMZ284 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6201) AUTHORS Jacobs JZ, Ciccaglione KM, Tournier V, Zaratiegui M TITLE Implementation of the CRISPR-Cas9 system in fission yeast. JOURNAL Nat Commun. 2014 Oct 29;5:5344. doi: 10.1038/ncomms6344. PUBMED 25352017 REFERENCE 2 (bases 1 to 6201) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6201 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(29..51) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 151..170 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" rep_origin 539..1320 /label=ars1 /note="Schizosaccharomyces pombe autonomously replicating sequence ars1" CDS 1930..2724 /codon_start=1 /gene="Schizosaccharomyces pombe ura4" /product="orotidine 5'-phosphate decarboxylase, required for uracil biosynthesis" /label=S. pombe ura4 /translation="MDARVFQSYSARAEGMKNPIAKELLALMEEKQSNLSVAVDLTKKS EILELVDKIGPYVCVIKTHIDVVEDFDQDMVEKLVALGKKHRFLIFEDRKFADIGNTVK LQYASGVYKIASWAHITNCHTVPGEGIIQGLKEVGLPLGRGLLLLAEMSSKGSLATGSY TEKTLEWFEKHTDFCFGFIAGRRFPNLQSDYITMSPGIGLDVKGDGLGQQYRTPEEVIV NCGSDIIIVGRGVYGAGRNPVVEAKRYREAGWKAYQQRLSQH" primer_bind 3350..3372 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 3364..3381 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 3365..3381 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" misc_RNA complement(3431..3482) /label=sTRSV HHRz /note="hammerhead ribozyme from the tobacco ringspot virus satellite RNA (Khvorova et al., 2003)" promoter complement(3587..3943) /label=rrk1 promoter/leader /note="promoter and leader RNA for the Schizosaccharomyces pombe rrk1 gene encoding the K-RNA component of RNase P (Jacobs et al., 2014)" primer_bind complement(3980..3996) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3980..3996) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(3993..4015) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4004..4020 /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(4028..4058) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4073..4094 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(4211..4228) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4382..4970) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4462..4481) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5141..6001) /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 5764..5783 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6002..6106) /gene="bla" /label=AmpR promoter primer_bind 6174..6192 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" ORIGIN 1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 181 accataatcg atggccaacc ttccaattca ttaaatcgag tcctccatca gatgatttac 241 aaggttttgc atagaatccc taagtaagag aagttgttag tgatttcttc aagtttacta 301 aaatttacat acaaatggat gatctcttct ccattgtttc ctttcttctt gcaaacgcgt 361 tttacaaaga gatgacatgc tattttctga ttattttttt ctatagtttt ctattttgta 421 cgtccttggg agtccgaaat gtaaaatcgg ggtatggtta gtagcggcag cgttcattat 481 ggtatataaa agataaaaag taggggcaag cgagacagaa tgggatacaa gggcatcgtc 541 tataattata gctaaaaatt gtattttaat ttgtattttt tgtaatttta tttctcattg 601 ttttacttaa aatgaatgcg aattagagaa aacttcaacg aaatgtcaaa ataagctcag 661 caaaatatac aattttaggg aaagcgatca gcaacttatt ctccgttgta gattcttttt 721 cttcataaat tccaacaatg taactgccaa ttgatttttc tctagatgct aataaattga 781 aactaatgaa tataataaca caggttttga taaattgtaa actcttactc aaaaaaagaa 841 tgttaaaact aatttaacct ttttataaag gctcatggcg ttaaagagga tagtaaaaat 901 tctacaattt tttaatttaa tgtcacaatt gtcttgcgaa tataattcta ttaattttcg 961 ctaactgctg ttttagtact attactttca atatgcctta tatgcaactt taattcatga 1021 agaatgcaat atttttgcaa tctttcttgt actgagcgct ttttatcatt tagtactcat 1081 tatttaattt ttcagtaaag ggcaataaat aaatttgtag aagatgcaat gtaatctcct 1141 ctatcctttt gctcatatgt ttatgagtat acctagtcta gaaaggcttg tatttaaaat 1201 attattcaat aaaaattcac aatttttacg acatgtgcta ccattcactt aacttcctga 1261 ttataaaatt ggttcgttta tactaattac ttaagtacct ttaactaaac aaaatgccta 1321 tatatatatt aatttacaat gagtgtcaga taagtcacta tgtccgagtg gttaaggagt 1381 tagactcgcc atcgatgagc ttagctacaa atcccactgg ctatatgtat gcatttgtgt 1441 taaaaaagtt tgtatagatt atttaatcta ctcagcattc tttctctaaa taggaatttg 1501 ttacttaatg gagaaaaaaa tgtttcgatt tacctagtgt atttgtttgt atactcacgt 1561 ttaatttcaa acatccattc tatcttgtgt aatttttggc atggtgaaaa agataatcag 1621 ccttataatc tttacaaaag taagaaattc tgtaaataag ccttaatgcc cttgctttaa 1681 attaaaatgg ttctttttca tgataatgtt tgcactttgt gaatatattt tagatagttc 1741 tgtgaggtat aattaagatg ttttagagac ttatacaatt ttgtctttat aaattcttaa 1801 ttgattttac catcccagtt taactatgct tcgtcggcat ctctgcacat gtcgtgtttt 1861 cttaccgtat tgtcctacca agaacctctt ttttgcttgg atcgaaatta aaggtttaaa 1921 agcaaagtta tggatgctag agtatttcaa agctattcag ctagagctga ggggatgaaa 1981 aatcccattg ccaaggaatt gttggctttg atggaagaaa agcaaagcaa cttgtcagtc 2041 gcggtcgatt tgacgaagaa atccgaaatc ttagaattgg tagataaaat tggaccctat 2101 gtctgtgtta tcaagacaca tattgacgtt gtcgaggatt tcgaccagga tatggtagaa 2161 aaactggtgg ccttaggtaa aaagcatcgt tttcttatct ttgaggatcg caaattcgca 2221 gacattggaa ataccgtcaa gctacaatat gcatctggtg tgtacaaaat tgcttcttgg 2281 gctcatatca caaattgcca tacagtgcca ggcgagggta ttatacaagg cctcaaagaa 2341 gttggtttac ctttgggacg tggtctcttg cttttggctg aaatgtcttc caaaggctct 2401 ttggctactg gttcctacac agagaaaacc ttagaatggt ttgagaagca taccgatttt 2461 tgctttggct ttatagctgg tcgtcgattt cctaaccttc aaagcgacta cataactatg 2521 tcccctggta tcggcttgga tgttaaagga gacgggctgg gacagcaata tcgtactcct 2581 gaagaagtga ttgtaaactg cggtagcgat atcatcattg ttggtcgtgg agtctatgga 2641 gctggtcgta atcctgttgt cgaagccaag agatatagag aagctggttg gaaggcatat 2701 cagcaaagac tttctcagca ttaaaaaaag actaatgtaa aatttttttg gttggttatt 2761 gaaaaagtcg atgccttgtt tgcgtttgtt ttcctaggcg ttttatgtca gaaggcattt 2821 agaattagta tacaagtact ctttggtaaa attttatgta gcgactaaaa tattaactat 2881 tatagataaa caccttggga ataaaaagta atttgctata gtaatttatt aaacatgctc 2941 ctacaacatt accacaatct tttctcttgg attgacattg aataagaaaa gagtgaattt 3001 ttttagactt gtaatgataa ctatgtacaa agccaatgaa agatgtatgt agatgaatgt 3061 aaaataccat gtagacaaac aagataaaac ttggttataa acattggtgt tggaacagaa 3121 taaattagat gtcaaaaagt ttcgtcaata tcacaagctc atcgcgatgg tatgcggtgt 3181 gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgccattc gccattcagg 3241 ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg 3301 aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga 3361 cgttgtaaaa cgacggccag tgccaagctt gcatgcctgc aggtcgactc tagaggatcc 3421 ccacggtacc cctgtttcgt cctcacggac tcatcagacc ggaaagcaca tccggtgaca 3481 gggcaccacc gactcggtgc cactcacttt tttcaagttg ataacggact agccttattt 3541 taacttgcta tttctagctc taaaaccgag gcatctgaac aatagattct tcggtacagg 3601 ttatgttttt tggcaacagc aactattcaa atcaactgaa agttttttag aattcaacaa 3661 cttccaatac tcaagtgtcc aagctagcaa actgtttaat aatcgtgacc caacattcct 3721 ttcatgcgca ccctcagcgt cacataaatc aacactttac aacacctatt caaagcttct 3781 atatgccttc gtcatttaaa tgtctaacat ttcaaaaaag tgtttgctaa tggtaaattc 3841 ttgagttttc ttggaagcac atatataacg agtgtgactg ttctacgctc cttgctgact 3901 agtcatcagt ctacgcatgc caactaccac caacataagc aaaaggtacc cggggtaccg 3961 agctcgaatt cgtaatcatg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa 4021 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga 4081 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt 4141 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct 4201 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat 4261 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga 4321 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt 4381 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt 4441 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc 4501 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa 4561 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct 4621 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta 4681 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg 4741 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc 4801 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta 4861 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg 4921 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt 4981 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg 5041 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta 5101 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg 5161 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg 5221 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc 5281 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg 5341 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg 5401 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag 5461 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat 5521 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc 5581 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc 5641 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa 5701 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac 5761 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt 5821 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc 5881 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa 5941 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca 6001 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat 6061 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa 6121 aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc 6181 gtatcacgag gccctttcgt c //