LOCUS       sequence-368415-        3644 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_zeo 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 3644)
  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 3644)
  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_zeo
FEATURES             Location/Qualifiers
     source          1..3644
                     /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"
     terminator      complement(549..746)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     promoter        complement(1139..1482)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     primer_bind     complement(1514..1533)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        1557..1825
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     primer_bind     complement(1887..1906)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1888..1906)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             complement(2017..2676)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     2591..2610
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(2677..2779)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     rep_origin      2880..3468
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3369..3388
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3622..3639
                     /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 atcatgagat gcctgcaagc aattcgttct
      781 gtatcaggcg caggagcgtc ccgtccgggt cgaccaaagc ggccatcgtg cctccccact
      841 cctgcagttc gggggcatgg atgcgcggat agccgctgct ggtttcctgg atgccgacgg
      901 atttgcactg ccggtagaac tccgcgaggt cgtccagcct caggcagcag ctgaaccaac
      961 tcgcgagggg atcgagcccg gggtgggcga agaactccag catgagatcc ccgcgctgga
     1021 ggatcatcca gccggcgtcc cggaaaacga ttccgaagcc caacctttca tagaaggcgg
     1081 cggtgggatc gaaatctcgt gatggcaggt tgggcgtcgc ttggtcggtc atacccatgg
     1141 ttgtttatgt tcggatgtga tgtgagaact gtatcctagc aagattttaa aaggaagtat
     1201 atgaaagaag aacctcagtg gcaaatccta accttttata tttctctaca ggggcgcggc
     1261 gtggggacaa ttcaacgcgt ctgtgagggg agcgtttccc tgctcgcagg tctgcagcga
     1321 ggagccgtaa tttttgcttc gcgccgtgcg gccatcaaaa tgtatggatg caaatgatta
     1381 tacatgggga tgtatgggct aaatgtatgg gcgacagtca catcatgccc ctgagctgcg
     1441 cacgtcaaga ctgtcaagga gggtattctg ggcctccatg tcgctgatta ccctgttatc
     1501 cctacactct cagtacaatc tgctctgatg ccgcatagtt aagccagccg ggtaattctt
     1561 tgaaaagata atgtatgatt atgctttcac tcatatttat acagaaactt gatgttttct
     1621 ttcgagtata tacaaggtga ttacatgtac gtttgaagta caactctaga ttttgtagtg
     1681 ccctcttggg ctagcggtaa aggtgcgcat tttttcacac cctacaatgt tctgttcaaa
     1741 agattttggt caaacgctgt agaagtgaaa gttggtgcgc atgtttcggc gttcgaaact
     1801 tctccgcagt gaaagataaa tgatcccatt cctgtagact tcttaattaa gacgtcagaa
     1861 ttctcgaggc ggccgcatgt gagtctccct atagtgagtc gtattaattt cgcgggcgga
     1921 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagtagcacc
     1981 aggcgtttaa gggcaccaat aactgcctta aaaaaattac gccccgccct gccactcatc
     2041 gcagtactgt tgtaattcat taagcattct gccgacatgg aagccatcac aaacggcatg
     2101 atgaacctga atcgccagcg gcatcagcac cttgtcgcct tgcgtataat atttgcccat
     2161 ggtgaaaacg ggggcgaaga agttgtccat attggccacg tttaaatcaa aactggtgaa
     2221 actcacccag ggattggctg agacaaaaaa catattctca ataaaccctt tagggaaata
     2281 ggccaggttt tcaccgtaac acgccacatc ttgcgaatat atgtgtagaa actgccggaa
     2341 atcgtcgtgg tattcactcc agagcgatga aaacgtttca gtttgctcat ggaaaacggt
     2401 gtaacaaggg tgaacactat cccatatcac cagctcaccg tctttcattg ccatacgaaa
     2461 ttccggatga gcattcatca ggcgggcaag aatgtgaata aaggccggat aaaacttgtg
     2521 cttatttttc tttacggtct ttaaaaaggc cgtaatatcc agctgaacgg tctggttata
     2581 ggtacattga gcaactgact gaaatgcctc aaaatgttct ttacgatgcc attgggatat
     2641 atcaacggtg gtatatccag tgattttttt ctccatttta gcttccttag ctcctgaaaa
     2701 tctcgataac tcaaaaaata cgcccggtag tgatcttatt tcattatggt gaaagttgga
     2761 acctcttacg tgccgatcaa agtctcattt tcgccaaaag ttgtcatgac caaaatccct
     2821 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     2881 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     2941 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     3001 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
     3061 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     3121 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     3181 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     3241 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     3301 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     3361 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     3421 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaat
     3481 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     3541 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     3601 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagc
//
