LOCUS Exported 8908 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION sgRNA with 1x MS2, Pol II promoter with ribozyme-gRNA-ribozyme design for yeast cells. ACCESSION . VERSION . KEYWORDS pJZC625 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8908) 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 8908) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8908 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 245..461 /gene="S. cerevisiae URA3" /label=URA3 promoter promoter 1625..2329 /gene="S. cerevisiae ADH1" /label=ADH1 promoter /note="promoter for alcohol dehydrogenase 1" misc_RNA 2417..2492 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" misc_RNA 2497..2515 /label=MS2 stem loop /note="stem loop that binds the bacteriophage MS2 coat protein" promoter 3128..3471 /label=TEF promoter /note="Ashbya gossypii TEF promoter" terminator 4290..4487 /label=TEF terminator /note="Ashbya gossypii TEF terminator" primer_bind complement(4916..4935) /label=T7 /note="T7 promoter, forward primer" promoter complement(4917..4935) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(4942..4959) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(4942..4958) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4951..4973) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 5099..5554 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(5186..5205) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 5396..5417 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(5910..5929) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 6029..6051 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(6089..6107) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" misc_feature 6171..6674 /label=CEN/ARS /note="S. cerevisiae CEN6 centromere fused to an autonomously replicating sequence" promoter 6731..6835 /gene="bla" /label=AmpR promoter CDS 6836..7696 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(7054..7073) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 7867..8455 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 8356..8375 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 8609..8626 /label=L4440 /note="L4440 vector, forward primer" protein_bind 8743..8764 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 8779..8809 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 8817..8833 /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 8822..8844 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 8841..8857 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 8841..8857 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" promoter 8878..8896 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" ORIGIN 1 ggccggccat ttaaatgttt aaacctactg cgccaattga tgacaataca gacgatgata 61 acaaaccgaa gttatctgat gtagaaaagg attaaagatg ctaagagata gtgatgatat 121 ttcataaata atgtaattct atatatgtta attacctttt ttgcgaggca tatttatggt 181 gaaggataag ttttgaccat caaagaaggt taatgtggct gtggtttcag ggtccataaa 241 gcttttcaat tcatcttttt tttttttgtt cttttttttg attccggttt ctttgaaatt 301 tttttgattc ggtaatctcc gagcagaagg aagaacgaag gaaggagcac agacttagat 361 tggtatatat acgcatatgt ggtgttgaag aaacatgaaa ttgcccagta ttcttaaccc 421 aactgcacag aacaaaaacc tgcaggaaac gaagataaat catgtcgaaa gctacatata 481 aggaacgtgc tgctactcat cctagtcctg ttgctgccaa gctatttaat atcatgcacg 541 aaaagcaaac aaacttgtgt gcttcattgg atgttcgtac caccaaggaa ttactggagt 601 tagttgaagc attaggtccc aaaatttgtt tactaaaaac acatgtggat atcttgactg 661 atttttccat ggagggcaca gttaagccgc taaaggcatt atccgccaag tacaattttt 721 tactcttcga agacagaaaa tttgctgaca ttggtaatac agtcaaattg cagtactctg 781 cgggtgtata cagaatagca gaatgggcag acattacgaa tgcacacggt gtggtgggcc 841 ctaaaacaag aagagggttg actacatcac gatgaggggg atcgaagaaa tgatggtaaa 901 tgaaatagga aatcaaggag catgaaggca aaagacaaat ataagggtcg aacgaaaaat 961 aaagtgaaaa gtgttgatat gatgtatttg gctttgcggc gccgaaaaaa cgagtttacg 1021 caattgcaca atcatgctga ctctgtggcg gacccgcgct cttgccggcc cggcgataac 1081 gctgggcgtg aggctgtgcc cggcggagtt ttttgcgcct gcattttcca aggtttaccc 1141 tgcgctaagg ggcgagattg gagaagcaat aagaatgccg gttggggttg cgatgatgac 1201 gaccacgaca actggtgtca ttatttaagt tgccgaaaga acctgagtgc atttgcaaca 1261 tgagtatact agaagaatga gccaagactt gcgagacgcg agtttgccgg tggtgcgaac 1321 aatagagcga ccatgacctt gaaggtgaga cgcgcataac cgctagagta ctttgaagag 1381 gaaacagcaa tagggttgct accagtataa atagacaggt acatacaaca ctggaaatgg 1441 ttgtctgttt gagtacgctt tcaattcatt tgggtgtgca ctttattatg ttacaatatg 1501 gaagggaact ttacacttct cctatgcaca tatattaatt aaagtccaat gctagtagag 1561 aaggggggta acacccctcc gcgctctttt ccgatttttt tctaaaccgt ggaatatttc 1621 ggatatcctt ttgttgtttc cgggtgtaca atatggactt cctcttttct ggcaaccaaa 1681 cccatacatc gggattccta taataccttc gttggtctcc ctaacatgta ggtggcggag 1741 gggagatata caatagaaca gataccagac aggacataat gggctaaaca agactacacc 1801 aattacactg cctcattgat ggtggtacat aacgaactaa tactgtagcc ctagacttga 1861 tagccatcat catatcgaag tttcactacc ctttttccat ttgccatcta ttgaagtaat 1921 aataggcgca tgcaacttct tttctttttt tttcttttct ctctcccccg ttgttgtctc 1981 accatatccg caatgacaaa aaaatgatgg aagacactaa aggaaaaaat taacgacaaa 2041 gacagcacca acagatgtcg ttgttccaga gctgatgagg ggtatctcga agcacacgaa 2101 actttttcct tccttcattc acgcacacta ctctctaatg agcaacggta tacggccttc 2161 cttccagtta cttgaatttg aaataaaaaa aagtttgctg tcttgctatc aagtataaat 2221 agacctgcaa ttattaatct tttgtttcct cgtcattgtt ctcgttccct ttcttccttg 2281 tttctttttc tgcacaatat ttcaagctat accaagcata caatcaacta tctcatatac 2341 aatgtctctc gagaaaagtc tgatgagtcc gtgaggacga aacgagtaag ctcgtcactt 2401 ttctctatca ctgatagttt tagagctaga aatagcaagt taaaataagg ctagtccgtt 2461 atcaacttga aaaagtggca ccgagtcggt gcgcgcacat gaggatcacc catgtgcttt 2521 tggccggcat ggtcccagcc tcctcgctgg cgccggctgg gcaacatgct tcggcatggc 2581 gaatgggacg cggccgccac cgcggtggag ctctaagcaa atagctaaat tatatacgaa 2641 ttaatattat gattaagtgt ttacgtgagt gcgatatttt tattactatc ttatacagtt 2701 gtatatactc tataaaatga gttgtctatt aattaacgcg atgaatgctt tctgggttta 2761 cctctccaac aactctagtt tacttctcaa tacattcaat tgtatttgat ttgtcaatac 2821 ttcatcatta atcaattcta tagttttgtt tttctcgttt atttccaaat ttaatgcatc 2881 aattttatta ttcaatttgt cgttgatttt ggttaatgat tttatggttt gatctctggc 2941 attgattgtt tgtgttagtt tttcattatt gataattaaa ttatttaagt tagttatcaa 3001 ctcggtgttt tcaagtttca agttttcaat ttctttagag tttattagat ttgtcaaagt 3061 ttctgaattg cttgattggt ccagatctgt ttagcttgcc tcgtccccgc cgggtcaccc 3121 ggccagcgac atggaggccc agaataccct ccttgacagt cttgacgtgc gcagctcagg 3181 ggcatgatgt gactgtcgcc cgtacattta gcccatacat ccccatgtat aatcatttgc 3241 atccatacat tttgatggcc gcacggcgcg aagcaaaaat tacggctcct cgctgcagac 3301 ctgcgagcag ggaaacgctc ccctcacaga cgcgttgaat tgtccccacg ccgcgcccct 3361 gtagagaaat ataaaaggtt aggatttgcc actgaggttc ttctttcata tacttccttt 3421 taaaatcttg ctaggataca gttctcacat cacatccgaa cataaacaac catgacagtc 3481 aacactaaga cctatagtga gagagcagaa actcatgcct caccagtagc acagcgatta 3541 tttcgattaa tggaactgaa gaaaaccaat ttatgtgcat caattgacgt tgataccact 3601 aaggaattcc ttgaattaat tgataaatta ggtccttatg tatgcttaat caagactcat 3661 attgatataa tcaatgattt ttcctatgaa tccactattg aaccattatt agaactttca 3721 cgtaaacatc aatttatgat ttttgaagat agaaaatttg ctgatattgg taataccgta 3781 aagaaacaat atattggtgg agtttataaa attagtagtt gggcagatat taccaatgct 3841 catggtgtca ctgggaatgg agtggttgaa ggattaaaac agggagctaa agaaaccacc 3901 accaaccaag agccaagagg gttattgatg ttagctgaat tatcatcagt gggatcatta 3961 gcatatggag aatattctca aaaaactgtt gaaattgcta aatccgataa ggaatttgtt 4021 attggattta ttgcccaacg tgatatgggt ggccaagaag aaggatttga ttggcttatt 4081 atgacacctg gagttggatt agatgataaa ggtgatggat taggacaaca atatagaact 4141 gttgatgaag ttgttagcac tggaactgat attatcattg ttggtagagg attgtttggt 4201 aaaggaagag atccagatat tgaaggtaaa aggtatagaa atgctggttg gaatgcttat 4261 ttgaaaaaga ctggccaatt ataatcagta ctgacaataa aaagattctt gttttcaaga 4321 acttgtcatt tgtatagttt ttttatattg tagttgttct attttaatca aatgttagcg 4381 tgatttatat tttttttcgc ctcgacatca tctgcccaga tgcgaagtta agtgcgcaga 4441 aagtaatatc atgcgtcaat cgtatgtgaa tgctggtcgc tatactgctg tcgattcgat 4501 actaacgccg ccatccagtg tcgaaaacga gctcgaattc atcgacccgg gaatctcggt 4561 cgtaatgatt tctataatga cgaaaaaaaa aaaattggaa agaaaaagct tcatggcctt 4621 tataaaaagg aactatccaa tacctcgcca gaaccaagta acagtatttt acggggcaca 4681 aatcaagaac aataagacag gactgtaaag atggacgcat tgaactccaa agaacaacaa 4741 gagttccaaa aagtagtgga acaaaagcaa atgaaggatt tcatgcgttt gtactctaat 4801 ctggtagaaa gatgtttcac agactgtgtc aatgacttca caacatcaaa gctaaccaat 4861 aaggaacaaa catgcatcat gaagtgcgtt taaacggcca cgctggccca attcgcccta 4921 tagtgagtcg tattacaatt cactggccgt cgttttacaa cgtcgtgact gggaaaaccc 4981 tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag 5041 cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggac 5101 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct 5161 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg 5221 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt 5281 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca 5341 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga 5401 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa 5461 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac 5521 gcgaatttta acaaaatatt aacgcttaca atttcctgat gcggtatttt ctccttacgc 5581 atctgtgcgg tatttcacac cgcatagggt aataactgat ataattaaat tgaagctcta 5641 atttgtgagt ttagtattag gatctctacc cttggcgaaa agtcctctgc caacaatgat 5701 gatatctgat ccaccactta caacttcgtc gacggttctg tactgctgac ccaatatgcg 5761 tatatatacc aatctaagtc tgtgctcctt ccttcgttct tccttctgtt cggagattac 5821 cgaatcaaaa aaatttcaag gaaaccgaaa tcaaaaaaaa gaataaaaaa aaaatgatga 5881 attgaaaagg tggtatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc 5941 cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca 6001 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg 6061 tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat 6121 gtcatgataa taatggtttc ttagacgtcg gcgcgccctg ggtccttttc atcacgtgct 6181 ataaaaataa ttataattta aattttttaa tataaatata taaattaaaa atagaaagta 6241 aaaaaagaaa ttaaagaaaa aatagttttt gttttccgaa gatgtaaaag actctagggg 6301 gatcgccaac aaatactacc ttttatcttg ctcttcctgc tctcaggtat taatgccgaa 6361 ttgtttcatc ttgtctgtgt agaagaccac acacgaaaat cctgtgattt tacattttac 6421 ttatcgttaa tcgaatgtat atctatttaa tctgcttttc ttgtctaata aatatatatg 6481 taaagtacgc tttttgttga aattttttaa acctttgttt attttttttt cttcattccg 6541 taactcttct accttcttta tttactttct aaaatccaaa tacaaaacat aaaaataaat 6601 aaacacagag taaattccca aattattcca tcattaaaag atacgaggcg cgtgtaagtt 6661 acaggcaagc gatccgtcct aagaaaccag gcgcgccagg acgtcaggtg gcacttttcg 6721 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc 6781 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag 6841 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt 6901 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt 6961 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga 7021 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat 7081 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga 7141 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag 7201 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg 7261 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg 7321 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt 7381 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg 7441 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc 7501 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg 7561 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac 7621 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact 7681 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa 7741 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa 7801 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg 7861 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc 7921 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac 7981 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca 8041 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt 8101 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc 8161 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg 8221 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc 8281 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac 8341 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct 8401 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc 8461 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt 8521 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac 8581 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg 8641 cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga 8701 caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac 8761 tcattaggca ccccaggctt tacactttat gcttccggct cctatgttgt gtggaattgt 8821 gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca agctcggaat 8881 taaccctcac taaagggaac aaaagctg //