LOCUS Exported 5289 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION EasyClone-MarkerFree guiding RNA vector to direct Cas9 to cut at site XII-2. ACCESSION . VERSION . KEYWORDS pCfB3048(gRNA XII-2) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5289) AUTHORS Jessop-Fabre MM, Jakociunas T, Stovicek V, Dai Z, Jensen MK, Keasling JD, Borodina I TITLE EasyClone-MarkerFree: A vector toolkit for marker-less integration of genes into Saccharomyces cerevisiae via CRISPR-Cas9. JOURNAL Biotechnol J. 2016 Aug;11(8):1110-7. doi: 10.1002/biot.201600147. Epub 2016 Jun 23. PUBMED 27166612 REFERENCE 2 (bases 1 to 5289) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5289 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(29..51) /label=pGEX 3' /note="pGEX vectors, reverse primer" gene 175..1294 /label=natMX6 /note="yeast selectable marker conferring nourseothricin resistance" promoter 175..518 /label=TEF promoter /note="Ashbya gossypii TEF promoter" CDS 519..1091 /codon_start=1 /gene="Streptomyces noursei nat1" /product="nourseothricin acetyltransferase" /label=NrsR /note="confers resistance to nourseothricin" /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTTTGDGF TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT LCGLDTALYDGTASDGEQALYMSMPCP" terminator 1097..1294 /label=TEF terminator /note="Ashbya gossypii TEF terminator" promoter 1468..1736 /label=SNR52 promoter /note="promoter for the S. cerevisiae small nucleolar RNA gene SNR52" misc_RNA 1757..1831 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" terminator 1836..1855 /label=SUP4 terminator /note="transcription terminator for the S. cerevisiae SUP4 tRNA gene" primer_bind complement(1957..1974) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(2128..2716) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(2208..2227) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(2887..3747) /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 3510..3529 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(3748..3852) /gene="bla" /label=AmpR promoter rep_origin 3879..5221 /label=2u ori /note="yeast 2u plasmid origin of replication" protein_bind complement(4842..4889) /label=FRT /bound_moiety="FLP recombinase from the Saccharomyces cerevisiae 2u plasmid" /note="FLP-mediated recombination occurs in the 8-bp core sequence TCTAGAAA (Turan and Bode, 2011)." primer_bind 5262..5280 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" ORIGIN 1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggct 121 gcatttaaat gatctgttta gcttgcctcg tccccgccgg gtcacccggc cagcgacatg 181 gaggcccaga ataccctcct tgacagtctt gacgtgcgca gctcaggggc atgatgtgac 241 tgtcgcccgt acatttagcc catacatccc catgtataat catttgcatc catacatttt 301 gatggccgca cggcgcgaag caaaaattac ggctcctcgc tgcagacctg cgagcaggga 361 aacgctcccc tcacagacgc gttgaattgt ccccacgccg cgcccctgta gagaaatata 421 aaaggttagg atttgccact gaggttcttc tttcatatac ttccttttaa aatcttgcta 481 ggatacagtt ctcacatcac atccgaacat aaacaaccat gggtaccact cttgacgaca 541 cggcttaccg gtaccgcacc agtgtcccgg gggacgccga ggccatcgag gcactggatg 601 ggtccttcac caccgacacc gttttccgcg tcaccaccac cggggacggc ttcaccctgc 661 gggaggtgcc ggtggacccg cccctgacca aggtgttccc cgacgacgaa tcggacgacg 721 aatcggacga cggggaggac ggcgacccgg actcccggac gttcgtcgcg tacggggacg 781 acggcgacct ggcgggcttc gtggtcgtct cgtactccgg ctggaaccgc cggctgaccg 841 tcgaggacat cgaggtcgcc ccggagcacc gggggcacgg ggtcgggcgc gcgttgatgg 901 ggctcgcgac ggagttcgcc cgcgagcggg gcgccgggca cctctggctg gaggtcacca 961 acgtcaacgc accggcgatc cacgcgtacc ggcggatggg gttcaccctc tgcggcctgg 1021 acaccgccct gtacgacggc accgcctcgg acggcgagca ggcgctctac atgagcatgc 1081 cctgccccta atcagtactg acaataaaaa gattcttgtt ttcaagaact tgtcatttgt 1141 atagtttttt tatattgtag ttgttctatt ttaatcaaat gttagcgtga tttatatttt 1201 ttttcgcctc gacatcatct gcccagatgc gaagttaagt gcgcagaaag taatatcatg 1261 cgtcaatcgt atgtgaatgc tggtcgctat actgctgtcg attcgatact aacgccgcca 1321 tccagtgtcg aaaacgagct gcggccgcct acagggcgcg tccattcgcc attcaggctg 1381 cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggaatgc 1441 gtgcgatagg gaacaaaagc tggagcttct ttgaaaagat aatgtatgat tatgctttca 1501 ctcatattta tacagaaact tgatgttttc tttcgagtat atacaaggtg attacatgta 1561 cgtttgaagt acaactctag attttgtagt gccctcttgg gctagcggta aaggtgcgca 1621 ttttttcaca ccctacaatg ttctgttcaa aagattttgg tcaaacgctg tagaagtgaa 1681 agttggtgcg catgtttcgg cgttcgaaac ttctccgcag tgaaagataa atgatctgaa 1741 actctaatcc tactatgttt tagagctaga aatagcaagt taaaataagg ctagtccgtt 1801 atcaacttga aaaagtggca ccgagtcggt ggtgcttttt ttgtttttta tgtcttcgag 1861 tcatgtaatt agttaatcgc gtgcattcca gctgcattaa tgaatcggcc aacgcgcggg 1921 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc 1981 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac 2041 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa 2101 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca 2161 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc 2221 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata 2281 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta 2341 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca 2401 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga 2461 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg 2521 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg 2581 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg 2641 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag 2701 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa 2761 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat 2821 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc 2881 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc 2941 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc 3001 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc 3061 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc 3121 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt 3181 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc 3241 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa 3301 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt 3361 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg 3421 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc 3481 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa 3541 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt 3601 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt 3661 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag 3721 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta 3781 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat 3841 aggggttccg cgcacatttc cccgaaaagt gccacctgaa cgaagcatct gtgcttcatt 3901 ttgtagaaca aaaatgcaac gcgagagcgc taatttttca aacaaagaat ctgagctgca 3961 tttttacaga acagaaatgc aacgcgaaag cgctatttta ccaacgaaga atctgtgctt 4021 catttttgta aaacaaaaat gcaacgcgag agcgctaatt tttcaaacaa agaatctgag 4081 ctgcattttt acagaacaga aatgcaacgc gagagcgcta ttttaccaac aaagaatcta 4141 tacttctttt ttgttctaca aaaatgcatc ccgagagcgc tatttttcta acaaagcatc 4201 ttagattact ttttttctcc tttgtgcgct ctataatgca gtctcttgat aactttttgc 4261 actgtaggtc cgttaaggtt agaagaaggc tactttggtg tctattttct cttccataaa 4321 aaaagcctga ctccacttcc cgcgtttact gattactagc gaagctgcgg gtgcattttt 4381 tcaagataaa ggcatccccg attatattct ataccgatgt ggattgcgca tactttgtga 4441 acagaaagtg atagcgttga tgattcttca ttggtcagaa aattatgaac ggtttcttct 4501 attttgtctc tatatactac gtataggaaa tgtttacatt ttcgtattgt tttcgattca 4561 ctctatgaat agttcttact acaatttttt tgtctaaaga gtaatactag agataaacat 4621 aaaaaatgta gaggtcgagt ttagatgcaa gttcaaggag cgaaaggtgg atgggtaggt 4681 tatataggga tatagcacag agatatatag caaagagata cttttgagca atgtttgtgg 4741 aagcggtatt cgcaatattt tagtagctcg ttacagtccg gtgcgttttt ggttttttga 4801 aagtgcgtct tcagagcgct tttggttttc aaaagcgctc tgaagttcct atactttcta 4861 gagaatagga acttcggaat aggaacttca aagcgtttcc gaaaacgagc gcttccgaaa 4921 atgcaacgcg agctgcgcac atacagctca ctgttcacgt cgcacctata tctgcgtgtt 4981 gcctgtatat atatatacat gagaagaacg gcatagtgcg tgtttatgct taaatgcgta 5041 cttatatgcg tctatttatg taggatgaaa ggtagtctag tacctcctgt gatattatcc 5101 cattccatgc ggggtatcgt atgcttcctt cagcactacc ctttagctgt tctatatgct 5161 gccactcctc aattggatta gtctcatcct tcaatgctat catttccttt gatattggat 5221 catactaaga aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc 5281 cctttcgtc //