LOCUS Exported 5268 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION sgRNA with MS2-PP7 for yeast cells. ACCESSION . VERSION . KEYWORDS pJZC593 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5268) 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 5268) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5268 /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 546..565 /label=SUP4 terminator /note="transcription terminator for the S. cerevisiae SUP4 tRNA gene" promoter complement(594..612) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(633..649) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(633..649) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(646..668) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 657..673 /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(681..711) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 726..747 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(864..881) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(1035..1623) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(1794..2654) /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 2417..2436 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(2655..2759) /gene="bla" /label=AmpR promoter misc_feature 2796..3299 /label=CEN/ARS /note="S. cerevisiae CEN6 centromere fused to an autonomously replicating sequence" primer_bind 3341..3359 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind complement(3397..3419) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 3519..3538 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" promoter 3563..3778 /gene="S. cerevisiae URA3" /label=URA3 promoter CDS 3779..4582 /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(4713..5168) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4850..4871) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 5062..5081 /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 tgccacgagt aaggagttta tatggaaacc cttactcgtg 541 ttcccttttt ttgtttttta tgtctctgca gagttcggta ccagcttttg ttccctttag 601 tgagggttaa tttcgagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt 661 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt 721 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg 781 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttt 841 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg 901 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat 961 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc 1021 gcgttgctgg cgtttttcca taggctcggc ccccctgacg agcatcacaa aaatcgacgc 1081 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt cccccctgga 1141 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt 1201 ctcccttcgg gaagcgtggc gctttctcaa tgctcacgct gtaggtatct cagttcggtg 1261 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc 1321 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg 1381 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc 1441 ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg 1501 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc 1561 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct 1621 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt 1681 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa 1741 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa 1801 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc 1861 tgactgcccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct 1921 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca 1981 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt 2041 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt 2101 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc 2161 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgaaaaaa agcggttagc 2221 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt 2281 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact 2341 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc 2401 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt 2461 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg 2521 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct 2581 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa 2641 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt 2701 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc 2761 acatttcccc gaaaagtgcc acctgggtcc ttttcatcac gtgctataaa aataattata 2821 atttaaattt tttaatataa atatataaat taaaaataga aagtaaaaaa agaaattaaa 2881 gaaaaaatag tttttgtttt ccgaagatgt aaaagactct agggggatcg ccaacaaata 2941 ctacctttta tcttgctctt cctgctctca ggtattaatg ccgaattgtt tcatcttgtc 3001 tgtgtagaag accacacacg aaaatcctgt gattttacat tttacttatc gttaatcgaa 3061 tgtatatcta tttaatctgc ttttcttgtc taataaatat atatgtaaag tacgcttttt 3121 gttgaaattt tttaaacctt tgtttatttt tttttcttca ttccgtaact cttctacctt 3181 ctttatttac tttctaaaat ccaaatacaa aacataaaaa taaataaaca cagagtaaat 3241 tcccaaatta ttccatcatt aaaagatacg aggcgcgtgt aagttacagg caagcgatcc 3301 gtcctaagaa accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc 3361 ctttcgtctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga 3421 gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc 3481 agcgggtgtt ggcgggtgtc ggggctggct taactatgcg gcatcagagc agattgtact 3541 gagagtgcac cacgcttttc aattcaattc atcatttttt ttttattctt ttttttgatt 3601 tcggtttcct tgaaattttt ttgattcggt aatctccgaa cagaaggaag aacgaaggaa 3661 ggagcacaga cttagattgg tatatatacg catatgtagt gttgaagaaa catgaaattg 3721 cccagtattc ttaacccaac tgcacagaac aaaaacgtgc aggaaacgaa gataaatcat 3781 gtcgaaagct acatataagg aacgtgctgc tactcatcct agtcctgttg ctgccaagct 3841 atttaatatc atgcacgaaa agcaaacaaa cttgtgtgct tcattggatg ttcgtaccac 3901 caaggaatta ctggagttag ttgaagcatt aggtcccaaa atttgtttac taaaaacaca 3961 tgtggatatc ttgactgatt tttccatgga gggcacagtt aagccgctaa aggcattatc 4021 cgccaagtac aattttttac tcttcgaaga cagaaaattt gctgacattg gtaatacagt 4081 caaattgcag tactctgcgg gtgtatacag aatagcagaa tgggcagaca ttacgaatgc 4141 acacggtgtg gtgggcccag gtattgttag cggtttgaag caggcggcag aagaagtaac 4201 aaaggaacct agaggccttt tgatgttagc agaattgtca tgcaagggct ccctatctac 4261 tggagaatat actaagggta ctgttgacat tgcgaagagc gacaaagatt ttgttatcgg 4321 ctttattgct caaagagaca tgggtggaag agatgaaggt tacgattggt tgattatgac 4381 acccggtgtg ggtttagatg acaagggaga cgcattgggt caacagtata gaaccgtgga 4441 tgatgtggtc tctacaggat ctgacattat tattgttgga agaggactat ttgcaaaggg 4501 aagggatgct aaggtagagg gtgaacgtta cagaaaagca ggctgggaag catatttgag 4561 aagatgcggc cagcaaaact aaaaaactgt attataagta aatgcatgta tactaaactc 4621 acaaattaga gcttcaattt aattatatca gttattaccc tatgcggtgt gaaataccgc 4681 acagatgcgt aaggagaaaa taccgcatca ggaaattgta aacgttaata ttttgttaaa 4741 attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa 4801 aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa 4861 caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca 4921 gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg 4981 taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc 5041 ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc 5101 aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca 5161 gggcgcgtcg cgccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg 5221 ggcctcttcg ctattacgcc agctggcgaa ggggggatgt gctgcaag //