LOCUS Exported 5711 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Multi-copy (2 micron) yeast plasmid containing a functional NatMX marker cassette conferring resistance against nourseothricin. Recipient plasmid for sgRNA or crRNA expression cassettes.. ACCESSION . VERSION . KEYWORDS pRN1120 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5711) AUTHORS Verwaal R, Buiting-Wiessenhaan N, Dalhuijsen S, Roubos JA TITLE CRISPR/Cpf1 enables fast and simple genome editing of Saccharomyces cerevisiae. JOURNAL Yeast. 2017 Sep 8. doi: 10.1002/yea.3278. PUBMED 28886218 REFERENCE 2 (bases 1 to 5711) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5711 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(525..544) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 644..666 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(704..722) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 790..894 /gene="bla" /label=AmpR promoter CDS 895..1755 /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(1113..1132) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 1926..2514 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 2415..2434 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 2668..2685 /label=L4440 /note="L4440 vector, forward primer" protein_bind 2802..2823 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 2838..2868 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 2876..2892 /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 2881..2903 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 2900..2916 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 2900..2916 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" promoter 2937..2955 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind 2983..2999 /label=pBluescriptKS /note="For pBluescript vector" primer_bind 2984..3000 /label=KS primer /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3034..3050) /label=SK primer /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3034..3050) /label=pBluescriptSK /note="For pBluescript vector" rep_origin 3665..4542 /label=2u ori /note="yeast 2u plasmid origin of replication" promoter 4600..4943 /label=TEF promoter /note="Ashbya gossypii TEF promoter" CDS 4951..5520 /codon_start=1 /gene="Streptomyces noursei nat1" /product="nourseothricin acetyltransferase" /label=NrsR /note="confers resistance to nourseothricin" /translation="MTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGFT LREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNRR LTVEDIEVAPEHRGHGVGRTLMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFTL CGLDTALYDGTASDGEQALYMSMPCP" terminator 5522..8 /label=TEF terminator /note="Ashbya gossypii TEF terminator" ORIGIN 1 ctatactgct gtcgattcga tactaacgcc gccatccagt gtcgacggat cctaggtgta 61 cataaacttt ataaatgaaa ttcataatag aaacgacacg aaattacaaa atggaatatg 121 ttcatagggt agacgaaact atatacgcaa tctacataca tttatcaaga aggagaaaaa 181 ggaggatgta aaggaataca ggtaagcaaa ttgatactaa tggctcaacg tgataaggaa 241 aaagaattgc actttaacat taatattgac aaggaggagg gcaccacaca aaaagttagg 301 tgtaacagaa aatcatgaaa ctatgattcc taatttatat attggaggat tttctctaaa 361 aaaaaaaaaa tacaacaaat aaaaaacact caatgacctg accatttgat ggagtttaag 421 tcaatacctt cttgaaccat ttcccataat ggtgaaagtt ccctcaagaa ttttactctg 481 tcagaaacgg ccttaacgac gtagtcgata tggtgcactc tcagtacaat ctgctctgat 541 gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct 601 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt 661 cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta 721 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg 781 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg 841 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt 901 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt 961 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg 1021 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa 1081 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt 1141 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag 1201 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt 1261 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga 1321 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt 1381 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta 1441 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg 1501 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc 1561 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt 1621 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg 1681 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg 1741 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa 1801 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa 1861 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga 1921 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg 1981 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact 2041 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac 2101 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg 2161 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg 2221 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga 2281 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc 2341 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg 2401 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc 2461 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc 2521 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt 2581 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc 2641 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc 2701 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac 2761 aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact 2821 cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg 2881 agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa gctcgaaatt 2941 aaccctcact aaagggaaca aaagctggta ccgggccccc cctcgaggtc gacggtatcg 3001 ataagcttga tatcgaattc ctgcagcccg ggggatccac tagttctaga gcggccgcat 3061 gctagctccg gattatcgat gataagctgt caaacatgag aattaattcc acggactata 3121 gactatacct agtatactcc gtctactgta cgatacactt ccgctcaggt ccttgtcctt 3181 taacgaggcc ttaccactct tttgttactc tattgatcca gctcagcaaa ggcagtgtga 3241 tctaagattc tatcttcgcg atgtagtaaa actagctaga ccgagaaaga gactagaaat 3301 gcaaaaggca cttctacaat ggctgccatc attattatcc gatgtgacgc tgcagcttct 3361 caatgatatt cgaatacgct ttgaggagat acagcctaat atccgacaaa ctgttttaca 3421 gatttacgat cgtacttgtt acccatcatt gaattttgaa catccgaacc tgggagtttt 3481 ccctgaaaca gatagtatat ttgaacctgt ataataatat atagtctagc gctttacgga 3541 agacaatgta tgtatttcgg ttcctggaga aactattgca tctattgcat aggtaatctt 3601 gcacgtcgca tccccggttc attttctgcg tttccatctt gcacttcaat agcatatctt 3661 tgttaacgaa gcatctgtgc ttcattttgt agaacaaaaa tgcaacgcga gagcgctaat 3721 ttttcaaaca aagaatctga gctgcatttt tacagaacag aaatgcaacg cgaaagcgct 3781 attttaccaa cgaagaatct gtgcttcatt tttgtaaaac aaaaatgcaa cgcgagagcg 3841 ctaatttttc aaacaaagaa tctgagctgc atttttacag aacagaaatg caacgcgaga 3901 gcgctatttt accaacaaag aatctatact tcttttttgt tctacaaaaa tgcatcccga 3961 gagcgctatt tttctaacaa agcatcttag attacttttt ttctcctttg tgcgctctat 4021 aatgcagtct cttgataact ttttgcactg taggtccgtt aaggttagaa gaaggctact 4081 ttggtgtcta ttttctcttc cataaaaaaa gcctgactcc acttcccgcg tttactgatt 4141 actagcgaag ctgcgggtgc attttttcaa gataaaggca tccccgatta tattctatac 4201 cgatgtggat tgcgcatact ttgtgaacag aaagtgatag cgttgatgat tcttcattgg 4261 tcagaaaatt atgaacggtt tcttctattt tgtctctata tactacgtat aggaaatgtt 4321 tacattttcg tattgttttc gattcactct atgaatagtt cttactacaa tttttttgtc 4381 taaagagtaa tactagagat aaacataaaa aatgtagagg tcgagtttag atgcaagttc 4441 aaggagcgaa aggtggatgg gtaggttata tagggatata gcacagagat atatagcaaa 4501 gagatacttt tgagcaatgt ttgtggaagc ggtattcgca atgccggagc tcgtacgttc 4561 gaacttaagg cctcgtcccc gccgggtcac ccggccagcg acatggaggc ccagaatacc 4621 ctccttgaca gtcttgacgt gcgcagctca ggggcatgat gtgactgtcg cccgtacatt 4681 tagcccatac atccccatgt ataatcattt gcatccatac attttgatgg ccgcacggcg 4741 cgaagcaaaa attacggctc ctcgctgcag acctgcgagc agggaaacgc tcccctcaca 4801 gacgcgttga attgtcccca cgccgcgccc ctgtagagaa atataaaagg ttaggatttg 4861 ccactgaggt tcttctttca tatacttcct tttaaaatct tgctaggata cagttctcac 4921 atcacatccg aacataaaca accatgtaaa atgaccactc ttgacgacac ggcttaccgg 4981 taccgcacca gtgtcccggg ggacgccgag gccatcgagg cactggatgg gtccttcacc 5041 accgacaccg tcttccgcgt caccgccacc ggggacggct tcaccctgcg ggaggtgccg 5101 gtggacccgc ccctgaccaa ggtgttcccc gacgacgaat cggacgacga atcggacgac 5161 ggggaggacg gcgacccgga ctcccggacg ttcgtcgcgt acggggacga cggcgacctg 5221 gcgggcttcg tggtcgtctc gtactccggc tggaaccgcc ggctgaccgt cgaggacatc 5281 gaggtcgccc cggagcaccg ggggcacggg gtcgggcgca cgttgatggg gctcgcgacg 5341 gagttcgccc gcgagcgggg cgccgggcac ctctggctgg aggtcaccaa cgtcaacgca 5401 ccggcgatcc acgcgtaccg gcggatgggg ttcaccctct gcggcctgga caccgccctg 5461 tacgacggca ccgcctcgga cggcgagcag gcgctctaca tgagcatgcc ctgcccctag 5521 tactgacaat aaaaagattc ttgttttcaa gaacttgtca tttgtatagt ttttttatat 5581 tgtagttgtt ctattttaat caaatgttag cgtgatttat attttttttc gcctcgacat 5641 catctgccca gatgcgaagt taagtgcgca gaaagtaata tcatgcgtca atcgtatgtg 5701 aatgctggtc g //