LOCUS Exported 5220 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION sgRNA with 1x MS2 for yeast cells. ACCESSION . VERSION . KEYWORDS pJZC545 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5220) AUTHORS Zalatan JG, Lee ME, Almeida R, Gilbert LA, Whitehead EH, La Russa M, Tsai JC, Weissman JS, Dueber JE, Qi LS, Lim WA TITLE Engineering Complex Synthetic Transcriptional Programs with CRISPR RNA Scaffolds. JOURNAL Cell. 2014 Dec 18. pii: S0092-8674(14)01570-0. doi: 10.1016/j.cell.2014.11.052. PUBMED 25533786 REFERENCE 2 (bases 1 to 5220) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5220 /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..476 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" misc_RNA 481..499 /label=MS2 stem loop /note="stem loop that binds the bacteriophage MS2 coat protein" terminator 502..521 /label=SUP4 terminator /note="transcription terminator for the S. cerevisiae SUP4 tRNA gene" promoter complement(550..568) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(589..605) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(589..605) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(602..624) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 613..629 /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(637..667) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 682..703 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(820..837) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(991..1579) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(1750..2610) /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 2373..2392 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(2611..2715) /gene="bla" /label=AmpR promoter misc_feature 2752..3255 /label=CEN/ARS /note="S. cerevisiae CEN6 centromere fused to an autonomously replicating sequence" primer_bind 3297..3315 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind complement(3353..3375) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 3475..3494 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" promoter 3519..3734 /gene="S. cerevisiae URA3" /label=URA3 promoter CDS 3735..4538 /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(4669..5124) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4806..4827) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 5018..5037 /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 cggtgcgcgc 481 acatgaggat cacccatgtg ctttttttgt tttttatgtc tctgcagagt tcggtaccag 541 cttttgttcc ctttagtgag ggttaatttc gagcttggcg taatcatggt catagctgtt 601 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa 661 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact 721 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc 781 ggggagaggc ggttttcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg 841 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc 901 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag 961 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctcggccccc ctgacgagca 1021 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca 1081 ggcgttcccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg 1141 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcaatgct cacgctgtag 1201 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt 1261 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca 1321 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg 1381 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt 1441 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc 1501 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg 1561 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg 1621 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta 1681 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg 1741 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg 1801 ttcatccata gttgcctgac tgcccgtcgt gtagataact acgatacggg agggcttacc 1861 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc 1921 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc 1981 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag 2041 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat 2101 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg 2161 aaaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt 2221 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag 2281 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg 2341 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt 2401 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct 2461 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac 2521 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat 2581 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat 2641 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca 2701 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gggtcctttt catcacgtgc 2761 tataaaaata attataattt aaatttttta atataaatat ataaattaaa aatagaaagt 2821 aaaaaaagaa attaaagaaa aaatagtttt tgttttccga agatgtaaaa gactctaggg 2881 ggatcgccaa caaatactac cttttatctt gctcttcctg ctctcaggta ttaatgccga 2941 attgtttcat cttgtctgtg tagaagacca cacacgaaaa tcctgtgatt ttacatttta 3001 cttatcgtta atcgaatgta tatctattta atctgctttt cttgtctaat aaatatatat 3061 gtaaagtacg ctttttgttg aaatttttta aacctttgtt tatttttttt tcttcattcc 3121 gtaactcttc taccttcttt atttactttc taaaatccaa atacaaaaca taaaaataaa 3181 taaacacaga gtaaattccc aaattattcc atcattaaaa gatacgaggc gcgtgtaagt 3241 tacaggcaag cgatccgtcc taagaaacca ttattatcat gacattaacc tataaaaata 3301 ggcgtatcac gaggcccttt cgtctcgcgc gtttcggtga tgacggtgaa aacctctgac 3361 acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag 3421 cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac tatgcggcat 3481 cagagcagat tgtactgaga gtgcaccacg cttttcaatt caattcatca tttttttttt 3541 attctttttt ttgatttcgg tttccttgaa atttttttga ttcggtaatc tccgaacaga 3601 aggaagaacg aaggaaggag cacagactta gattggtata tatacgcata tgtagtgttg 3661 aagaaacatg aaattgccca gtattcttaa cccaactgca cagaacaaaa acgtgcagga 3721 aacgaagata aatcatgtcg aaagctacat ataaggaacg tgctgctact catcctagtc 3781 ctgttgctgc caagctattt aatatcatgc acgaaaagca aacaaacttg tgtgcttcat 3841 tggatgttcg taccaccaag gaattactgg agttagttga agcattaggt cccaaaattt 3901 gtttactaaa aacacatgtg gatatcttga ctgatttttc catggagggc acagttaagc 3961 cgctaaaggc attatccgcc aagtacaatt ttttactctt cgaagacaga aaatttgctg 4021 acattggtaa tacagtcaaa ttgcagtact ctgcgggtgt atacagaata gcagaatggg 4081 cagacattac gaatgcacac ggtgtggtgg gcccaggtat tgttagcggt ttgaagcagg 4141 cggcagaaga agtaacaaag gaacctagag gccttttgat gttagcagaa ttgtcatgca 4201 agggctccct atctactgga gaatatacta agggtactgt tgacattgcg aagagcgaca 4261 aagattttgt tatcggcttt attgctcaaa gagacatggg tggaagagat gaaggttacg 4321 attggttgat tatgacaccc ggtgtgggtt tagatgacaa gggagacgca ttgggtcaac 4381 agtatagaac cgtggatgat gtggtctcta caggatctga cattattatt gttggaagag 4441 gactatttgc aaagggaagg gatgctaagg tagagggtga acgttacaga aaagcaggct 4501 gggaagcata tttgagaaga tgcggccagc aaaactaaaa aactgtatta taagtaaatg 4561 catgtatact aaactcacaa attagagctt caatttaatt atatcagtta ttaccctatg 4621 cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggaa attgtaaacg 4681 ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat 4741 aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg 4801 ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc 4861 gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt 4921 tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag 4981 cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg 5041 gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc 5101 ttaatgcgcc gctacagggc gcgtccattc gccattcagg ctgcgcaact gttgggaagg 5161 gcgatcggtg cgggcctctt cgctattacg ccagctggcg aaagggggat gtgctgcaag //