LOCUS Exported 5255 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION sgRNA with 2x MS2 (wt+f6) for yeast cells. ACCESSION . VERSION . KEYWORDS pJZC588 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5255) 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 5255) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5255 /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 483..501 /label=MS2 stem loop /note="stem loop that binds the bacteriophage MS2 coat protein" terminator 533..552 /label=SUP4 terminator /note="transcription terminator for the S. cerevisiae SUP4 tRNA gene" promoter complement(581..599) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(620..636) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(620..636) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(633..655) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 644..660 /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(668..698) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 713..734 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(851..868) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(1022..1610) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(1781..2641) /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 2404..2423 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(2642..2746) /gene="bla" /label=AmpR promoter misc_feature 2783..3286 /label=CEN/ARS /note="S. cerevisiae CEN6 centromere fused to an autonomously replicating sequence" primer_bind 3328..3346 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind complement(3384..3406) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 3506..3525 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" promoter 3550..3765 /gene="S. cerevisiae URA3" /label=URA3 promoter CDS 3766..4569 /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(4700..5155) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4837..4858) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 5049..5068 /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 cggtgcggga 481 gcacatgagg atcacccatg tgcgactccc acagtcactg gggagtcttc cctttttttg 541 ttttttatgt ctctgcagag ttcggtacca gcttttgttc cctttagtga gggttaattt 601 cgagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa 661 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga 721 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt 781 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggttttcgt attgggcgct 841 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat 901 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga 961 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt 1021 ttttccatag gctcggcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt 1081 ggcgaaaccc gacaggacta taaagatacc aggcgttccc ccctggaagc tccctcgtgc 1141 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa 1201 gcgtggcgct ttctcaatgc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct 1261 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta 1321 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg 1381 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc 1441 ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta 1501 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg 1561 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt 1621 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg 1681 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta 1741 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg 1801 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctgcccgtcg 1861 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc 1921 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg 1981 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg 2041 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag 2101 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat 2161 caaggcgagt tacatgatcc cccatgttgt gaaaaaaagc ggttagctcc ttcggtcctc 2221 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc 2281 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa 2341 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac 2401 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt 2461 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc 2521 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa 2581 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca 2641 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat 2701 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa 2761 aagtgccacc tgggtccttt tcatcacgtg ctataaaaat aattataatt taaatttttt 2821 aatataaata tataaattaa aaatagaaag taaaaaaaga aattaaagaa aaaatagttt 2881 ttgttttccg aagatgtaaa agactctagg gggatcgcca acaaatacta ccttttatct 2941 tgctcttcct gctctcaggt attaatgccg aattgtttca tcttgtctgt gtagaagacc 3001 acacacgaaa atcctgtgat tttacatttt acttatcgtt aatcgaatgt atatctattt 3061 aatctgcttt tcttgtctaa taaatatata tgtaaagtac gctttttgtt gaaatttttt 3121 aaacctttgt ttattttttt ttcttcattc cgtaactctt ctaccttctt tatttacttt 3181 ctaaaatcca aatacaaaac ataaaaataa ataaacacag agtaaattcc caaattattc 3241 catcattaaa agatacgagg cgcgtgtaag ttacaggcaa gcgatccgtc ctaagaaacc 3301 attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg 3361 cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct 3421 tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc 3481 gggtgtcggg gctggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccac 3541 gcttttcaat tcaattcatc attttttttt tattcttttt tttgatttcg gtttccttga 3601 aatttttttg attcggtaat ctccgaacag aaggaagaac gaaggaagga gcacagactt 3661 agattggtat atatacgcat atgtagtgtt gaagaaacat gaaattgccc agtattctta 3721 acccaactgc acagaacaaa aacgtgcagg aaacgaagat aaatcatgtc gaaagctaca 3781 tataaggaac gtgctgctac tcatcctagt cctgttgctg ccaagctatt taatatcatg 3841 cacgaaaagc aaacaaactt gtgtgcttca ttggatgttc gtaccaccaa ggaattactg 3901 gagttagttg aagcattagg tcccaaaatt tgtttactaa aaacacatgt ggatatcttg 3961 actgattttt ccatggaggg cacagttaag ccgctaaagg cattatccgc caagtacaat 4021 tttttactct tcgaagacag aaaatttgct gacattggta atacagtcaa attgcagtac 4081 tctgcgggtg tatacagaat agcagaatgg gcagacatta cgaatgcaca cggtgtggtg 4141 ggcccaggta ttgttagcgg tttgaagcag gcggcagaag aagtaacaaa ggaacctaga 4201 ggccttttga tgttagcaga attgtcatgc aagggctccc tatctactgg agaatatact 4261 aagggtactg ttgacattgc gaagagcgac aaagattttg ttatcggctt tattgctcaa 4321 agagacatgg gtggaagaga tgaaggttac gattggttga ttatgacacc cggtgtgggt 4381 ttagatgaca agggagacgc attgggtcaa cagtatagaa ccgtggatga tgtggtctct 4441 acaggatctg acattattat tgttggaaga ggactatttg caaagggaag ggatgctaag 4501 gtagagggtg aacgttacag aaaagcaggc tgggaagcat atttgagaag atgcggccag 4561 caaaactaaa aaactgtatt ataagtaaat gcatgtatac taaactcaca aattagagct 4621 tcaatttaat tatatcagtt attaccctat gcggtgtgaa ataccgcaca gatgcgtaag 4681 gagaaaatac cgcatcagga aattgtaaac gttaatattt tgttaaaatt cgcgttaaat 4741 ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa 4801 tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta 4861 ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca 4921 ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat 4981 cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg 5041 agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc 5101 acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcgcgc 5161 cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta 5221 ttacgccagc tggcgaaggg gggatgtgct gcaag //