LOCUS Exported 5202 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Yeast CEN/ARS vector (Ura3) that contains sgRNA controlled by SNR 52 promoter, targeting endogenous TRE elements of pTET07 promoter. ACCESSION . VERSION . KEYWORDS pSNR52-sgTET SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5202) AUTHORS Gilbert LA, Larson MH, Morsut L, Liu Z, Brar GA, Torres SE, Stern-Ginossar N, Brandman O, Whitehead EH, Doudna JA, Lim WA, Weissman JS, Qi LS TITLE CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes. JOURNAL Cell. 2013 Jul 9. pii: S0092-8674(13)00826-X. doi: 10.1016/j.cell.2013.06.044. PUBMED 23849981 REFERENCE 2 (bases 1 to 5202) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5202 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 30..52 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 44..61 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 45..61 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind 68..87 /label=T7 /note="T7 promoter, forward primer" promoter 68..86 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" promoter 112..380 /label=SNR52 promoter /note="promoter for the S. cerevisiae small nucleolar RNA gene SNR52" misc_RNA 401..475 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" terminator 480..499 /label=SUP4 terminator /note="transcription terminator for the S. cerevisiae SUP4 tRNA gene" promoter complement(528..546) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(567..583) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(567..583) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(580..602) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 591..607 /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(615..645) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 660..681 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(798..815) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(969..1557) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(1728..2588) /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFFHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind 2351..2370 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(2589..2693) /gene="bla" /label=AmpR promoter misc_feature 2730..3233 /label=CEN/ARS /note="S. cerevisiae CEN6 centromere fused to an autonomously replicating sequence" primer_bind 3275..3293 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind complement(3331..3353) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 3453..3472 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" promoter 3497..3712 /gene="S. cerevisiae URA3" /label=URA3 promoter CDS 3713..4516 /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" rep_origin complement(4647..5102) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4784..4805) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 4996..5015 /label=F1ori-R /note="F1 origin, reverse primer" ORIGIN 1 gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa cgacggccag 61 tgaattgtaa tacgactcac tatagggcga attggagctc actcggaatt ctctttgaaa 121 agataatgta tgattatgct ttcactcata tttatacaga aacttgatgt tttctttcga 181 gtatatacaa ggtgattaca tgtacgtttg aagtacaact ctagattttg tagtgccctc 241 ttgggctagc ggtaaaggtg cgcatttttt cacaccctac aatgttctgt tcaaaagatt 301 ttggtcaaac gctgtagaag tgaaagttgg tgcgcatgtt tcggcgttcg aaacttctcc 361 gcagtgaaag ataaatgatc acttttctct atcactgata gttttagagc tagaaatagc 421 aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtggtgct 481 ttttttgttt tttatgtctc tgcagagttc ggtaccagct tttgttccct ttagtgaggg 541 ttaatttcga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg 601 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa 661 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac 721 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg ttttcgtatt 781 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga 841 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca 901 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg 961 ctggcgtttt tccataggct cggcccccct gacgagcatc acaaaaatcg acgctcaagt 1021 cagaggtggc gaaacccgac aggactataa agataccagg cgttcccccc tggaagctcc 1081 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct 1141 tcgggaagcg tggcgctttc tcaatgctca cgctgtaggt atctcagttc ggtgtaggtc 1201 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta 1261 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca 1321 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag 1381 tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag 1441 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt 1501 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa 1561 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg 1621 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga 1681 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta 1741 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactg 1801 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg 1861 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga 1921 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt 1981 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt 2041 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc 2101 caacgatcaa ggcgagttac atgatccccc atgttgtgaa aaaaagcggt tagctccttc 2161 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca 2221 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag 2281 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg 2341 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa 2401 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa 2461 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga 2521 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga 2581 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg 2641 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt 2701 ccccgaaaag tgccacctgg gtccttttca tcacgtgcta taaaaataat tataatttaa 2761 attttttaat ataaatatat aaattaaaaa tagaaagtaa aaaaagaaat taaagaaaaa 2821 atagtttttg ttttccgaag atgtaaaaga ctctaggggg atcgccaaca aatactacct 2881 tttatcttgc tcttcctgct ctcaggtatt aatgccgaat tgtttcatct tgtctgtgta 2941 gaagaccaca cacgaaaatc ctgtgatttt acattttact tatcgttaat cgaatgtata 3001 tctatttaat ctgcttttct tgtctaataa atatatatgt aaagtacgct ttttgttgaa 3061 attttttaaa cctttgttta tttttttttc ttcattccgt aactcttcta ccttctttat 3121 ttactttcta aaatccaaat acaaaacata aaaataaata aacacagagt aaattcccaa 3181 attattccat cattaaaaga tacgaggcgc gtgtaagtta caggcaagcg atccgtccta 3241 agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg 3301 tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt 3361 cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg 3421 tgttggcggg tgtcggggct ggcttaacta tgcggcatca gagcagattg tactgagagt 3481 gcaccacgct tttcaattca attcatcatt ttttttttat tctttttttt gatttcggtt 3541 tccttgaaat ttttttgatt cggtaatctc cgaacagaag gaagaacgaa ggaaggagca 3601 cagacttaga ttggtatata tacgcatatg tagtgttgaa gaaacatgaa attgcccagt 3661 attcttaacc caactgcaca gaacaaaaac gtgcaggaaa cgaagataaa tcatgtcgaa 3721 agctacatat aaggaacgtg ctgctactca tcctagtcct gttgctgcca agctatttaa 3781 tatcatgcac gaaaagcaaa caaacttgtg tgcttcattg gatgttcgta ccaccaagga 3841 attactggag ttagttgaag cattaggtcc caaaatttgt ttactaaaaa cacatgtgga 3901 tatcttgact gatttttcca tggagggcac agttaagccg ctaaaggcat tatccgccaa 3961 gtacaatttt ttactcttcg aagacagaaa atttgctgac attggtaata cagtcaaatt 4021 gcagtactct gcgggtgtat acagaatagc agaatgggca gacattacga atgcacacgg 4081 tgtggtgggc ccaggtattg ttagcggttt gaagcaggcg gcagaagaag taacaaagga 4141 acctagaggc cttttgatgt tagcagaatt gtcatgcaag ggctccctat ctactggaga 4201 atatactaag ggtactgttg acattgcgaa gagcgacaaa gattttgtta tcggctttat 4261 tgctcaaaga gacatgggtg gaagagatga aggttacgat tggttgatta tgacacccgg 4321 tgtgggttta gatgacaagg gagacgcatt gggtcaacag tatagaaccg tggatgatgt 4381 ggtctctaca ggatctgaca ttattattgt tggaagagga ctatttgcaa agggaaggga 4441 tgctaaggta gagggtgaac gttacagaaa agcaggctgg gaagcatatt tgagaagatg 4501 cggccagcaa aactaaaaaa ctgtattata agtaaatgca tgtatactaa actcacaaat 4561 tagagcttca atttaattat atcagttatt accctatgcg gtgtgaaata ccgcacagat 4621 gcgtaaggag aaaataccgc atcaggaaat tgtaaacgtt aatattttgt taaaattcgc 4681 gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc 4741 ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag 4801 tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga 4861 tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc 4921 actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa 4981 cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt 5041 agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc 5101 gtcgcgccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc 5161 ttcgctatta cgccagctgg cgaagggggg atgtgctgca ag //