LOCUS Exported 6263 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Encodes a gRNA that targets Can1.Y in yeast.. ACCESSION . VERSION . KEYWORDS p426-SNR52p-gRNA.CAN1.Y-SUP4t SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6263) AUTHORS Dicarlo JE, Norville JE, Mali P, Rios X, Aach J, Church GM. TITLE Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. JOURNAL Nucleic Acids Res PUBMED 23460208 REFERENCE 2 (bases 1 to 6263) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6263 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(10..28) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" rep_origin 69..1411 /label=2u ori /note="yeast 2u plasmid origin of replication" protein_bind 401..448 /label=FRT /bound_moiety="FLP recombinase from the Saccharomyces cerevisiae 2u plasmid" /note="FLP-mediated recombination occurs in the 8-bp core sequence TCTAGAAA (Turan and Bode, 2011)." promoter 1438..1542 /gene="bla" /label=AmpR promoter CDS 1543..2403 /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 complement(1761..1780) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 2574..3162 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 3063..3082 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 3316..3333 /label=L4440 /note="L4440 vector, forward primer" protein_bind 3450..3471 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 3486..3516 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 3524..3540 /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 3529..3551 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 3548..3564 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 3548..3564 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 3583..3603 /label=T3 /note="T3 promoter, forward primer" promoter 3585..3603 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" promoter 3621..3889 /label=SNR52 promoter /note="promoter for the S. cerevisiae small nucleolar RNA gene SNR52" misc_RNA 3910..3984 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" terminator 3989..4008 /label=SUP4 terminator /note="transcription terminator for the S. cerevisiae SUP4 tRNA gene" terminator 4014..4261 /gene="S. cerevisiae CYC1" /label=CYC1 terminator /note="transcription terminator for CYC1" primer_bind complement(4279..4298) /label=T7 /note="T7 promoter, forward primer" promoter complement(4280..4298) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(4308..4325) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(4308..4324) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4317..4339) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 4465..4920 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4552..4571) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 4762..4783 /label=F1ori-F /note="F1 origin, forward primer" CDS complement(5051..5854) /codon_start=1 /gene="S. cerevisiae URA3" /product="orotidine-5'-phosphate decarboxylase, required for uracil biosynthesis" /label=URA3 /note="yeast auxotrophic marker, counterselectable with 5-fluoroorotic acid (5-FOA)" /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN" promoter complement(5855..6070) /gene="S. cerevisiae URA3" /label=URA3 promoter primer_bind complement(6094..6113) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 6213..6235 /label=pGEX 3' /note="pGEX vectors, reverse primer" ORIGIN 1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt 61 cttagtatga tccaatatca aaggaaatga tagcattgaa ggatgagact aatccaattg 121 aggagtggca gcatatagaa cagctaaagg gtagtgctga aggaagcata cgataccccg 181 catggaatgg gataatatca caggaggtac tagactacct ttcatcctac ataaatagac 241 gcatataagt acgcatttaa gcataaacac gcactatgcc gttcttctca tgtatatata 301 tatacaggca acacgcagat ataggtgcga cgtgaacagt gagctgtatg tgcgcagctc 361 gcgttgcatt ttcggaagcg ctcgttttcg gaaacgcttt gaagttccta ttccgaagtt 421 cctattctct agaaagtata ggaacttcag agcgcttttg aaaaccaaaa gcgctctgaa 481 gacgcacttt caaaaaacca aaaacgcacc ggactgtaac gagctactaa aatattgcga 541 ataccgcttc cacaaacatt gctcaaaagt atctctttgc tatatatctc tgtgctatat 601 ccctatataa cctacccatc cacctttcgc tccttgaact tgcatctaaa ctcgacctct 661 acatttttta tgtttatctc tagtattact ctttagacaa aaaaattgta gtaagaacta 721 ttcatagagt gaatcgaaaa caatacgaaa atgtaaacat ttcctatacg tagtatatag 781 agacaaaata gaagaaaccg ttcataattt tctgaccaat gaagaatcat caacgctatc 841 actttctgtt cacaaagtat gcgcaatcca catcggtata gaatataatc ggggatgcct 901 ttatcttgaa aaaatgcacc cgcagcttcg ctagtaatca gtaaacgcgg gaagtggagt 961 caggcttttt ttatggaaga gaaaatagac accaaagtag ccttcttcta accttaacgg 1021 acctacagtg caaaaagtta tcaagagact gcattataga gcgcacaaag gagaaaaaaa 1081 gtaatctaag atgctttgtt agaaaaatag cgctctcggg atgcattttt gtagaacaaa 1141 aaagaagtat agattctttg ttggtaaaat agcgctctcg cgttgcattt ctgttctgta 1201 aaaatgcagc tcagattctt tgtttgaaaa attagcgctc tcgcgttgca tttttgtttt 1261 acaaaaatga agcacagatt cttcgttggt aaaatagcgc tttcgcgttg catttctgtt 1321 ctgtaaaaat gcagctcaga ttctttgttt gaaaaattag cgctctcgcg ttgcattttt 1381 gttctacaaa atgaagcaca gatgcttcgt tcaggtggca cttttcgggg aaatgtgcgc 1441 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa 1501 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc 1561 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa 1621 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa 1681 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg 1741 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa 1801 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc 1861 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc 1921 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta 1981 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag 2041 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca 2101 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata 2161 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc 2221 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca 2281 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca 2341 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg 2401 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa 2461 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt 2521 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat 2581 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg 2641 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga 2701 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac 2761 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt 2821 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag 2881 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc 2941 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag 3001 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca 3061 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt 3121 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc 3181 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc 3241 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc 3301 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa 3361 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac 3421 tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc 3481 caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa 3541 tttcacacag gaaacagcta tgaccatgat tacgccaagc gcgcaattaa ccctcactaa 3601 agggaacaaa agctggagct tctttgaaaa gataatgtat gattatgctt tcactcatat 3661 ttatacagaa acttgatgtt ttctttcgag tatatacaag gtgattacat gtacgtttga 3721 agtacaactc tagattttgt agtgccctct tgggctagcg gtaaaggtgc gcattttttc 3781 acaccctaca atgttctgtt caaaagattt tggtcaaacg ctgtagaagt gaaagttggt 3841 gcgcatgttt cggcgttcga aacttctccg cagtgaaaga taaatgatcg atacgttctc 3901 tatggaggag ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact 3961 tgaaaaagtg gcaccgagtc ggtggtgctt tttttgtttt ttatgtcttc gagtcatgta 4021 attagttatg tcacgcttac gttcacgccc tccccccaca tccgctctaa ccgaaaagga 4081 aggagttaga caacctgaag tctaggtccc tatttatttt tttatagtta tgttagtatt 4141 aagaacgtta tttatatttc aaatttttct tttttttctg tacagacgcg tgtacgcatg 4201 taacattata ctgaaaacct tgcttgagaa ggttttggga cgctcgaagg ctttaatttg 4261 cggccggtac ccaattcgcc ctatagtgag tcgtattacg cgcgctcact ggccgtcgtt 4321 ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat 4381 ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag 4441 ttgcgcagcc tgaatggcga atggacgcgc cctgtagcgg cgcattaagc gcggcgggtg 4501 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg 4561 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg 4621 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt 4681 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt 4741 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta 4801 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa 4861 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt 4921 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tagggtaata 4981 actgatataa ttaaattgaa gctctaattt gtgagtttag tatacatgca tttacttata 5041 atacagtttt ttagttttgc tggccgcatc ttctcaaata tgcttcccag cctgcttttc 5101 tgtaacgttc accctctacc ttagcatccc ttccctttgc aaatagtcct cttccaacaa 5161 taataatgtc agatcctgta gagaccacat catccacggt tctatactgt tgacccaatg 5221 cgtctccctt gtcatctaaa cccacaccgg gtgtcataat caaccaatcg taaccttcat 5281 ctcttccacc catgtctctt tgagcaataa agccgataac aaaatctttg tcgctcttcg 5341 caatgtcaac agtaccctta gtatattctc cagtagatag ggagcccttg catgacaatt 5401 ctgctaacat caaaaggcct ctaggttcct ttgttacttc ttctgccgcc tgcttcaaac 5461 cgctaacaat acctgggccc accacaccgt gtgcattcgt aatgtctgcc cattctgcta 5521 ttctgtatac acccgcagag tactgcaatt tgactgtatt accaatgtca gcaaattttc 5581 tgtcttcgaa gagtaaaaaa ttgtacttgg cggataatgc ctttagcggc ttaactgtgc 5641 cctccatgga aaaatcagtc aagatatcca catgtgtttt tagtaaacaa attttgggac 5701 ctaatgcttc aactaactcc agtaattcct tggtggtacg aacatccaat gaagcacaca 5761 agtttgtttg cttttcgtgc atgatattaa atagcttggc agcaacagga ctaggatgag 5821 tagcagcacg ttccttatat gtagctttcg acatgattta tcttcgtttc ctgcaggttt 5881 ttgttctgtg cagttgggtt aagaatactg ggcaatttca tgtttcttca acactacata 5941 tgcgtatata taccaatcta agtctgtgct ccttccttcg ttcttccttc tgttcggaga 6001 ttaccgaatc aaaaaaattt caaggaaacc gaaatcaaaa aaaagaataa aaaaaaaatg 6061 atgaattgaa aaggtggtat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt 6121 aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc 6181 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc 6241 accgtcatca ccgaaacgcg cga //