LOCUS Exported 6145 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION EasyClone-MarkerFree guiding RNA vector to direct Cas9 to cut at sites X-2, XI-5, and XII-4. ACCESSION . VERSION . KEYWORDS pCfB3053(gRNA X-2, XI-5, XII-4) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6145) 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 6145) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6145 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(29..51) /label=pGEX 3' /note="pGEX vectors, reverse primer" gene 171..1290 /label=natMX6 /note="yeast selectable marker conferring nourseothricin resistance" promoter 171..514 /label=TEF promoter /note="Ashbya gossypii TEF promoter" CDS 515..1087 /codon_start=1 /gene="Streptomyces noursei nat1" /product="nourseothricin acetyltransferase" /label=NrsR /note="confers resistance to nourseothricin" /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT LCGLDTALYDGTASDGEQALYMSMPCP" terminator 1093..1290 /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" promoter 1896..2164 /label=SNR52 promoter /note="promoter for the S. cerevisiae small nucleolar RNA gene SNR52" misc_RNA 2185..2259 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" terminator 2264..2283 /label=SUP4 terminator /note="transcription terminator for the S. cerevisiae SUP4 tRNA gene" promoter 2324..2592 /label=SNR52 promoter /note="promoter for the S. cerevisiae small nucleolar RNA gene SNR52" misc_RNA 2613..2687 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" terminator 2692..2711 /label=SUP4 terminator /note="transcription terminator for the S. cerevisiae SUP4 tRNA gene" primer_bind complement(2813..2830) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(2984..3572) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(3064..3083) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(3743..4603) /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 4366..4385 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(4604..4708) /gene="bla" /label=AmpR promoter rep_origin 4735..6077 /label=2u ori /note="yeast 2u plasmid origin of replication" protein_bind complement(5698..5745) /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 6118..6136 /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 tcagcgggat 121 ttaaatgatc tgtttagctt gcctcgtccc cgccgggtca cccggccagc gacatggagg 181 cccagaatac cctccttgac agtcttgacg tgcgcagctc aggggcatga tgtgactgtc 241 gcccgtacat ttagcccata catccccatg tataatcatt tgcatccata cattttgatg 301 gccgcacggc gcgaagcaaa aattacggct cctcgctgca gacctgcgag cagggaaacg 361 ctcccctcac agacgcgttg aattgtcccc acgccgcgcc cctgtagaga aatataaaag 421 gttaggattt gccactgagg ttcttctttc atatacttcc ttttaaaatc ttgctaggat 481 acagttctca catcacatcc gaacataaac aaccatgggt accactcttg acgacacggc 541 ttaccggtac cgcaccagtg tcccggggga cgccgaggcc atcgaggcac tggatgggtc 601 cttcaccacc gacaccgttt tccgcgtcac cgccaccggg gacggcttca ccctgcggga 661 ggtgccggtg gacccgcccc tgaccaaggt gttccccgac gacgaatcgg acgacgaatc 721 ggacgacggg gaggacggcg acccggactc ccggacgttc gtcgcgtacg gggacgacgg 781 cgacctggcg ggcttcgtgg tcgtctcgta ctccggctgg aaccgccggc tgaccgtcga 841 ggacatcgag gtcgccccgg agcaccgggg gcacggggtc gggcgcgcgt tgatggggct 901 cgcgacggag ttcgcccgcg agcggggcgc cgggcacctc tggctggagg tcaccaacgt 961 caacgcaccg gcgatccacg cgtaccggcg gatggggttc accctctgcg gcctggacac 1021 cgccctgtac gacggcaccg cctcggacgg cgagcaggcg ctctacatga gcatgccctg 1081 cccctaatca gtactgacaa taaaaagatt cttgttttca agaacttgtc atttgtatag 1141 tttttttata ttgtagttgt tctattttaa tcaaatgtta gcgtgattta tatttttttt 1201 cgcctcgaca tcatctgccc agatgcgaag ttaagtgcgc agaaagtaat atcatgcgtc 1261 aatcgtatgt gaatgctggt cgctatactg ctgtcgattc gatactaacg ccgccatcca 1321 gtgtcgaaaa cgagctgcgg ccgcctacag ggcgcgtcgc gccattcgcc 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 atgatcctct 1741 cgaagtggtc acgtgcgttt tagagctaga aatagcaagt taaaataagg ctagtccgtt 1801 atcaacttga aaaagtggca ccgagtcggt ggtgcttttt ttgtttttta tgtcttcgag 1861 tcatgtaatt agttaaggga acaaaagctg gagcttcttt gaaaagataa tgtatgatta 1921 tgctttcact catatttata cagaaacttg atgttttctt tcgagtatat acaaggtgat 1981 tacatgtacg tttgaagtac aactctagat tttgtagtgc cctcttgggc tagcggtaaa 2041 ggtgcgcatt ttttcacacc ctacaatgtt ctgttcaaaa gattttggtc aaacgctgta 2101 gaagtgaaag ttggtgcgca tgtttcggcg ttcgaaactt ctccgcagtg aaagataaat 2161 gatctgagaa tactgttgta aaacgtttta gagctagaaa tagcaagtta aaataaggct 2221 agtccgttat caacttgaaa aagtggcacc gagtcggtgg tgcttttttt gttttttatg 2281 tcttcgagtc atgtaattag ttaagggaac aaaagctgga gcttctttga aaagataatg 2341 tatgattatg ctttcactca tatttataca gaaacttgat gttttctttc gagtatatac 2401 aaggtgatta catgtacgtt tgaagtacaa ctctagattt tgtagtgccc tcttgggcta 2461 gcggtaaagg tgcgcatttt ttcacaccct acaatgttct gttcaaaaga ttttggtcaa 2521 acgctgtaga agtgaaagtt ggtgcgcatg tttcggcgtt cgaaacttct ccgcagtgaa 2581 agataaatga tcgcttcaag aattgagtaa acgttttaga gctagaaata gcaagttaaa 2641 ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtggtg ctttttttgt 2701 tttttatgtc ttcgagtcat gtaattagtt aatcgcgtgc attccagctg cattaatgaa 2761 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca 2821 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg 2881 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc 2941 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc 3001 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac 3061 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc 3121 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata 3181 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc 3241 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca 3301 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag 3361 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta 3421 gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg 3481 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc 3541 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt 3601 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa 3661 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat 3721 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga 3781 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac 3841 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg 3901 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg 3961 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt 4021 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct 4081 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat 4141 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta 4201 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca 4261 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat 4321 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac 4381 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa 4441 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt 4501 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg 4561 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat 4621 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt 4681 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgaacgaa 4741 gcatctgtgc ttcattttgt agaacaaaaa tgcaacgcga gagcgctaat ttttcaaaca 4801 aagaatctga gctgcatttt tacagaacag aaatgcaacg cgaaagcgct attttaccaa 4861 cgaagaatct gtgcttcatt tttgtaaaac aaaaatgcaa cgcgagagcg ctaatttttc 4921 aaacaaagaa tctgagctgc atttttacag aacagaaatg caacgcgaga gcgctatttt 4981 accaacaaag aatctatact tcttttttgt tctacaaaaa tgcatcccga gagcgctatt 5041 tttctaacaa agcatcttag attacttttt ttctcctttg tgcgctctat aatgcagtct 5101 cttgataact ttttgcactg taggtccgtt aaggttagaa gaaggctact ttggtgtcta 5161 ttttctcttc cataaaaaaa gcctgactcc acttcccgcg tttactgatt actagcgaag 5221 ctgcgggtgc attttttcaa gataaaggca tccccgatta tattctatac cgatgtggat 5281 tgcgcatact ttgtgaacag aaagtgatag cgttgatgat tcttcattgg tcagaaaatt 5341 atgaacggtt tcttctattt tgtctctata tactacgtat aggaaatgtt tacattttcg 5401 tattgttttc gattcactct atgaatagtt cttactacaa tttttttgtc taaagagtaa 5461 tactagagat aaacataaaa aatgtagagg tcgagtttag atgcaagttc aaggagcgaa 5521 aggtggatgg gtaggttata tagggatata gcacagagat atatagcaaa gagatacttt 5581 tgagcaatgt ttgtggaagc ggtattcgca atattttagt agctcgttac agtccggtgc 5641 gtttttggtt ttttgaaagt gcgtcttcag agcgcttttg gttttcaaaa gcgctctgaa 5701 gttcctatac tttctagaga ataggaactt cggaatagga acttcaaagc gtttccgaaa 5761 acgagcgctt ccgaaaatgc aacgcgagct gcgcacatac agctcactgt tcacgtcgca 5821 cctatatctg cgtgttgcct gtatatatat atacatgaga agaacggcat agtgcgtgtt 5881 tatgcttaaa tgcgtactta tatgcgtcta tttatgtagg atgaaaggta gtctagtacc 5941 tcctgtgata ttatcccatt ccatgcgggg tatcgtatgc ttccttcagc actacccttt 6001 agctgttcta tatgctgcca ctcctcaatt ggattagtct catccttcaa tgctatcatt 6061 tcctttgata ttggatcata ctaagaaacc attattatca tgacattaac ctataaaaat 6121 aggcgtatca cgaggccctt tcgtc //