LOCUS Exported 978 bp ds-DNA linear SYN 30-MAY-2021 DEFINITION leu2 deletion gRNA cassette carried by pRS42H. ACCESSION . VERSION . KEYWORDS gRNA-leu-HYB SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 978) AUTHORS Zhang GC, Kong II, Kim H, Liu JJ, Cate JH, Jin YS TITLE Construction of a quadruple auxotrophic mutant of an industrial polyploid saccharomyces cerevisiae strain by using RNA-guided Cas9 nuclease. JOURNAL Appl Environ Microbiol. 2014 Dec;80(24):7694-701. doi: 10.1128/AEM.02310-14. Epub 2014 Oct 3. PUBMED 25281382 REFERENCE 2 (bases 1 to 978) AUTHORS . TITLE Direct Submission JOURNAL Exported May 30, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..978 /organism="synthetic DNA construct" /mol_type="other DNA" protein_bind 95..116 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 131..161 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 169..185 /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 174..196 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 193..209 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 193..209 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 228..248 /label=T3 /note="T3 promoter, forward primer" promoter 230..248 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" promoter 267..535 /label=SNR52 promoter /note="promoter for the S. cerevisiae small nucleolar RNA gene SNR52" misc_RNA 556..630 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" terminator 635..654 /label=SUP4 terminator /note="transcription terminator for the S. cerevisiae SUP4 tRNA gene" primer_bind complement(668..687) /label=T7 /note="T7 promoter, forward primer" promoter complement(669..687) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(697..714) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(697..713) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(706..728) /label=M13/pUC Forward /note="In lacZ gene" primer_bind complement(942..961) /label=F1ori-R /note="F1 origin, reverse primer" ORIGIN 1 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc 61 ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg 121 caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat 181 aacaatttca cacaggaaac agctatgacc atgattacgc caagcgcgca attaaccctc 241 actaaaggga acaaaagctg gagctctctt tgaaaagata atgtatgatt atgctttcac 301 tcatatttat acagaaactt gatgttttct ttcgagtata tacaaggtga ttacatgtac 361 gtttgaagta caactctaga ttttgtagtg ccctcttggg ctagcggtaa aggtgcgcat 421 tttttcacac cctacaatgt tctgttcaaa agattttggt caaacgctgt agaagtgaaa 481 gttggtgcgc atgtttcggc gttcgaaact tctccgcagt gaaagataaa tgatcaatca 541 cagccgaagc cattagtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta 601 tcaacttgaa aaagtggcac cgagtcggtg gtgctttttt tgttttttat gtctggtacc 661 caattcgccc tatagtgagt cgtattacgc gcgctcactg gccgtcgttt tacaacgtcg 721 tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc 781 cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct 841 gaatggcgaa atggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac 901 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc 961 ttcctttctc gccacgtt //