LOCUS Exported 5285 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION EasyClone-MarkerFree guiding RNA vector to direct Cas9 to cut at site XI-2. ACCESSION . VERSION . KEYWORDS pCfB3044(gRNA XI-2) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5285) 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 5285) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5285 /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 1464..1732 /label=SNR52 promoter /note="promoter for the S. cerevisiae small nucleolar RNA gene SNR52" misc_RNA 1753..1827 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" terminator 1832..1851 /label=SUP4 terminator /note="transcription terminator for the S. cerevisiae SUP4 tRNA gene" primer_bind complement(1953..1970) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(2124..2712) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(2204..2223) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(2883..3743) /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 3506..3525 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(3744..3848) /gene="bla" /label=AmpR promoter rep_origin 3875..5217 /label=2u ori /note="yeast 2u plasmid origin of replication" protein_bind complement(4838..4885) /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 5258..5276 /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 ggcgcgtcca ttcgccattc aggctgcgca 1381 actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg gaatgcgtgc 1441 gatagggaac aaaagctgga gcttctttga aaagataatg tatgattatg ctttcactca 1501 tatttataca gaaacttgat gttttctttc gagtatatac aaggtgatta catgtacgtt 1561 tgaagtacaa ctctagattt tgtagtgccc tcttgggcta gcggtaaagg tgcgcatttt 1621 ttcacaccct acaatgttct gttcaaaaga ttttggtcaa acgctgtaga agtgaaagtt 1681 ggtgcgcatg tttcggcgtt cgaaacttct ccgcagtgaa agataaatga tcgttgacca 1741 gttgatcagt tggttttaga gctagaaata gcaagttaaa ataaggctag tccgttatca 1801 acttgaaaaa gtggcaccga gtcggtggtg ctttttttgt tttttatgtc ttcgagtcat 1861 gtaattagtt aatcgcgtgc attccagctg cattaatgaa tcggccaacg cgcggggaga 1921 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc 1981 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa 2041 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt 2101 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa 2161 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt 2221 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg 2281 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc 2341 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc 2401 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta 2461 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct 2521 acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc 2581 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa 2641 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa 2701 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa 2761 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt 2821 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac 2881 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc 2941 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc 3001 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata 3061 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc 3121 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc 3181 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca 3241 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa 3301 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca 3361 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt 3421 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt 3481 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg 3541 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga 3601 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc 3661 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg 3721 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag 3781 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg 3841 gttccgcgca catttccccg aaaagtgcca cctgaacgaa gcatctgtgc ttcattttgt 3901 agaacaaaaa tgcaacgcga gagcgctaat ttttcaaaca aagaatctga gctgcatttt 3961 tacagaacag aaatgcaacg cgaaagcgct attttaccaa cgaagaatct gtgcttcatt 4021 tttgtaaaac aaaaatgcaa cgcgagagcg ctaatttttc aaacaaagaa tctgagctgc 4081 atttttacag aacagaaatg caacgcgaga gcgctatttt accaacaaag aatctatact 4141 tcttttttgt tctacaaaaa tgcatcccga gagcgctatt tttctaacaa agcatcttag 4201 attacttttt ttctcctttg tgcgctctat aatgcagtct cttgataact ttttgcactg 4261 taggtccgtt aaggttagaa gaaggctact ttggtgtcta ttttctcttc cataaaaaaa 4321 gcctgactcc acttcccgcg tttactgatt actagcgaag ctgcgggtgc attttttcaa 4381 gataaaggca tccccgatta tattctatac cgatgtggat tgcgcatact ttgtgaacag 4441 aaagtgatag cgttgatgat tcttcattgg tcagaaaatt atgaacggtt tcttctattt 4501 tgtctctata tactacgtat aggaaatgtt tacattttcg tattgttttc gattcactct 4561 atgaatagtt cttactacaa tttttttgtc taaagagtaa tactagagat aaacataaaa 4621 aatgtagagg tcgagtttag atgcaagttc aaggagcgaa aggtggatgg gtaggttata 4681 tagggatata gcacagagat atatagcaaa gagatacttt tgagcaatgt ttgtggaagc 4741 ggtattcgca atattttagt agctcgttac agtccggtgc gtttttggtt ttttgaaagt 4801 gcgtcttcag agcgcttttg gttttcaaaa gcgctctgaa gttcctatac tttctagaga 4861 ataggaactt cggaatagga acttcaaagc gtttccgaaa acgagcgctt ccgaaaatgc 4921 aacgcgagct gcgcacatac agctcactgt tcacgtcgca cctatatctg cgtgttgcct 4981 gtatatatat atacatgaga agaacggcat agtgcgtgtt tatgcttaaa tgcgtactta 5041 tatgcgtcta tttatgtagg atgaaaggta gtctagtacc tcctgtgata ttatcccatt 5101 ccatgcgggg tatcgtatgc ttccttcagc actacccttt agctgttcta tatgctgcca 5161 ctcctcaatt ggattagtct catccttcaa tgctatcatt tcctttgata ttggatcata 5221 ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt 5281 tcgtc //