LOCUS       sequence-368408-        3830 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template to generate via PCR two gRNAs for expression in S. 
            cerevisiae. The PCR product from pEasyG3_nat recombines in vivo with
            a PCR product from pEasyG3_mic..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3830)
  AUTHORS   Jacobus AP, Barreto JA, de Bem LS, Menegon YA, Fier I, Bueno JGR, 
            Dos Santos LV, Gross J
  TITLE     EasyGuide Plasmids Support in Vivo Assembly of gRNAs for CRISPR/Cas9
            Applications in Saccharomyces cerevisiae.
  JOURNAL   ACS Synth Biol. 2022 Nov 18;11(11):3886-3891. doi: 
            10.1021/acssynbio.2c00348. Epub 2022 Oct 18.
  PUBMED    36257021
REFERENCE   2  (bases 1 to 3830)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1021/acssynbio.2c00348"; journalName: "ACS Synth Biol"; date: 
            "2022-11-18- 18"; volume: "11"; issue: "11"; pages: "3886-3891"
COMMENT     Sequence Label: pEasyG3_nat
FEATURES             Location/Qualifiers
     source          1..3830
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        254..272
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     254..271
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     misc_RNA        400..474
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      479..498
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     gene            complement(549..1668)
                     /label=natMX6
                     /note="yeast selectable marker conferring nourseothricin 
                     resistance"
     terminator      complement(549..746)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(752..1324)
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     promoter        complement(1325..1668)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     primer_bind     complement(1700..1719)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        1743..2011
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     primer_bind     complement(2073..2092)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2074..2092)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             complement(2203..2862)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     2777..2796
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(2863..2965)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     rep_origin      3066..3654
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3555..3574
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3808..3825
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 cgaaaaatca ataatcagac aacaagatgt gcgaactcga tattttacac gactctcttt
       61 accaattctg ccccgaatta cacttaaaac gactcaacag cttaacgttg gcttgccacg
      121 cattacttga ctgtaaaact ctcactctta ccgaacttgg ccgtaacctg ccaaccaaag
      181 cgagaacaaa acataacatc aaacgaatcg accgattgtt aggtaatcgt cacctgcagg
      241 aaggtttaaa cgcatttagg tgacactata gaagtgtgta tcgctcgagg gatccgaatt
      301 cgaagtcttg gtacggaggt ccagatagcc cagtagctga cattcatccg gggtcagcac
      361 cgtttctgcg gactggcttt ctacgtgttc cgcttccttg ttttagagct agaaatagca
      421 agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtggtgctt
      481 tttttgtttt ttatgtctcg agagctcgtt ttcgacactg gatggcggcg ttagtatcga
      541 atcgacagca gtatagcgac cagcattcac atacgattga cgcatgatat tactttctgc
      601 gcacttaact tcgcatctgg gcagatgatg tcgaggcgaa aaaaaatata aatcacgcta
      661 acatttgatt aaaatagaac aactacaata taaaaaaact atacaaatga caagttcttg
      721 aaaacaagaa tctttttatt gtcagtactg attaggggca gggcatgctc atgtagagcg
      781 cctgctcgcc gtccgaggcg gtgccgtcgt acagggcggt gtccaggccg cagagggtga
      841 accccatccg ccggtacgcg tggatcgccg gtgcgttgac gttggtgacc tccagccaga
      901 ggtgcccggc gccccgctcg cgggcgaact ccgtcgcgag ccccatcaac gcgcgcccga
      961 ccccgtgccc ccggtgctcc ggggcgacct cgatgtcctc gacggtcagc cggcggttcc
     1021 agccggagta cgagacgacc acgaagcccg ccaggtcgcc gtcgtccccg tacgcgacga
     1081 acgtccggga gtccgggtcg ccgtcctccc cgtcgtccga ttcgtcgtcc gattcgtcgt
     1141 cggggaacac cttggtcagg ggcgggtcca ccggcacctc ccgcagggtg aagccgtccc
     1201 cggtggcggt gacgcggaag acggtgtcgg tggtgaagga cccatccagt gcctcgatgg
     1261 cctcggcgtc ccccgggaca ctggtgcggt accggtaagc cgtgtcgtca agagtggtac
     1321 ccatggttgt ttatgttcgg atgtgatgtg agaactgtat cctagcaaga ttttaaaagg
     1381 aagtatatga aagaagaacc tcagtggcaa atcctaacct tttatatttc tctacagggg
     1441 cgcggcgtgg ggacaattca acgcgtctgt gaggggagcg tttccctgct cgcaggtctg
     1501 cagcgaggag ccgtaatttt tgcttcgcgc cgtgcggcca tcaaaatgta tggatgcaaa
     1561 tgattataca tggggatgta tgggctaaat gtacgggcga cagtcacatc atgcccctga
     1621 gctgcgcacg tcaagactgt caaggagggt attctgggcc tccatgtcgc tgattaccct
     1681 gttatcccta cactctcagt acaatctgct ctgatgccgc atagttaagc cagccgggta
     1741 attctttgaa aagataatgt atgattatgc tttcactcat atttatacag aaacttgatg
     1801 ttttctttcg agtatataca aggtgattac atgtacgttt gaagtacaac tctagatttt
     1861 gtagtgccct cttgggctag cggtaaaggt gcgcattttt tcacacccta caatgttctg
     1921 ttcaaaagat tttggtcaaa cgctgtagaa gtgaaagttg gtgcgcatgt ttcggcgttc
     1981 gaaacttctc cgcagtgaaa gataaatgat cccattcctg tagacttctt aattaagacg
     2041 tcagaattct cgaggcggcc gcatgtgagt ctccctatag tgagtcgtat taatttcgcg
     2101 ggcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgagt
     2161 agcaccaggc gtttaagggc accaataact gccttaaaaa aattacgccc cgccctgcca
     2221 ctcatcgcag tactgttgta attcattaag cattctgccg acatggaagc catcacaaac
     2281 ggcatgatga acctgaatcg ccagcggcat cagcaccttg tcgccttgcg tataatattt
     2341 gcccatggtg aaaacggggg cgaagaagtt gtccatattg gccacgttta aatcaaaact
     2401 ggtgaaactc acccagggat tggctgagac aaaaaacata ttctcaataa accctttagg
     2461 gaaataggcc aggttttcac cgtaacacgc cacatcttgc gaatatatgt gtagaaactg
     2521 ccggaaatcg tcgtggtatt cactccagag cgatgaaaac gtttcagttt gctcatggaa
     2581 aacggtgtaa caagggtgaa cactatccca tatcaccagc tcaccgtctt tcattgccat
     2641 acgaaattcc ggatgagcat tcatcaggcg ggcaagaatg tgaataaagg ccggataaaa
     2701 cttgtgctta tttttcttta cggtctttaa aaaggccgta atatccagct gaacggtctg
     2761 gttataggta cattgagcaa ctgactgaaa tgcctcaaaa tgttctttac gatgccattg
     2821 ggatatatca acggtggtat atccagtgat ttttttctcc attttagctt ccttagctcc
     2881 tgaaaatctc gataactcaa aaaatacgcc cggtagtgat cttatttcat tatggtgaaa
     2941 gttggaacct cttacgtgcc gatcaaagtc tcattttcgc caaaagttgt catgaccaaa
     3001 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     3061 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     3121 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     3181 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     3241 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     3301 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     3361 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     3421 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     3481 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     3541 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     3601 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     3661 agcaatgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     3721 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     3781 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
//
