LOCUS       sequence-368411-        4286 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_hph 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 4286)
  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 4286)
  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_hph
FEATURES             Location/Qualifiers
     source          1..4286
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      174..371
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     terminator      complement(422..441)
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     misc_RNA        complement(446..520)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(648..666)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(649..666)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(925..942)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1096..1684)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1176..1195)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        1785..1887
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             1888..2547
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1954..1973)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     primer_bind     2658..2677
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2658..2676
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(2739..3007)
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     primer_bind     3031..3050
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     gene            join(3082..4286,1..371)
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     promoter        3082..3425
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             join(3426..4286,1..168)
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTSPRA
                     KE"
ORIGIN
        1 gcttgggcgc agggtcgatg cgacgcaatc gtccgatccg gagccgggac tgtcgggcgt
       61 acacaaatcg cccgcagaag cgcggccgtc tggaccgatg gctgtgtaga agtactcgcc
      121 gatagtggaa accgacgccc cagcactagt ccgagggcaa aggaataatc agtactgaca
      181 ataaaaagat tcttgttttc aagaacttgt catttgtata gtttttttat attgtagttg
      241 ttctatttta atcaaatgtt agcgtgattt atattttttt tcgcctcgac atcatctgcc
      301 cagatgcgaa gttaagtgcg cagaaagtaa tatcatgcgt caatcgtatg tgaatgctgg
      361 tcgctatact gctgtcgatt cgatactaac gccgccatcc agtgtccaaa acgagctctc
      421 gagacataaa aaacaaaaaa agcaccaccg actcggtgcc actttttcaa gttgataacg
      481 gactagcctt attttaactt gctatttcta gctctaaaac aaggaagcgg aacacgtaga
      541 aagccagtcc gcagaaacgg tgctgacccc ggatgaatgt cagctactgg gctatctgga
      601 cctccgtacc aagacttcga attcggatcc ctcgagcgat acacacttct atagtgtcac
      661 ctaaatgcgt ttaaaccttc ctgcaggtga cgattaccta acaatcggtc gattcgtttg
      721 atgttatgtt ttgttctcgc tttggttggc aggttacggc caagttcggt aagagtgaga
      781 gttttacagt caagtaatgc gtggcaagcc aacgttaagc tgttgagtcg ttttaagtgt
      841 aattcggggc agaattggta aagagagtcg tgtaaaatat cgagttcgca catcttgttg
      901 tctgattatt gatttttcgg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
      961 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     1021 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     1081 cgcattgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     1141 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     1201 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     1261 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     1321 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     1381 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     1441 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     1501 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     1561 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     1621 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     1681 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     1741 ttaagggatt ttggtcatga caacttttgg cgaaaatgag actttgatcg gcacgtaaga
     1801 ggttccaact ttcaccataa tgaaataaga tcactaccgg gcgtattttt tgagttatcg
     1861 agattttcag gagctaagga agctaaaatg gagaaaaaaa tcactggata taccaccgtt
     1921 gatatatccc aatggcatcg taaagaacat tttgaggcat ttcagtcagt tgctcaatgt
     1981 acctataacc agaccgttca gctggatatt acggcctttt taaagaccgt aaagaaaaat
     2041 aagcacaagt tttatccggc ctttattcac attcttgccc gcctgatgaa tgctcatccg
     2101 gaatttcgta tggcaatgaa agacggtgag ctggtgatat gggatagtgt tcacccttgt
     2161 tacaccgttt tccatgagca aactgaaacg ttttcatcgc tctggagtga ataccacgac
     2221 gatttccggc agtttctaca catatattcg caagatgtgg cgtgttacgg tgaaaacctg
     2281 gcctatttcc ctaaagggtt tattgagaat atgttttttg tctcagccaa tccctgggtg
     2341 agtttcacca gttttgattt aaacgtggcc aatatggaca acttcttcgc ccccgttttc
     2401 accatgggca aatattatac gcaaggcgac aaggtgctga tgccgctggc gattcaggtt
     2461 catcatgccg tttgtgatgg cttccatgtc ggcagaatgc ttaatgaatt acaacagtac
     2521 tgcgatgagt ggcagggcgg ggcgtaattt ttttaaggca gttattggtg cccttaaacg
     2581 cctggtgcta ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     2641 ggttccgccc gcgaaattaa tacgactcac tatagggaga ctcacatgcg gccgcctcga
     2701 gaattctgac gtcttaatta agaagtctac aggaatggga tcatttatct ttcactgcgg
     2761 agaagtttcg aacgccgaaa catgcgcacc aactttcact tctacagcgt ttgaccaaaa
     2821 tcttttgaac agaacattgt agggtgtgaa aaaatgcgca cctttaccgc tagcccaaga
     2881 gggcactaca aaatctagag ttgtacttca aacgtacatg taatcacctt gtatatactc
     2941 gaaagaaaac atcaagtttc tgtataaata tgagtgaaag cataatcata cattatcttt
     3001 tcaaagaatt acccggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgt
     3061 agggataaca gggtaatcag cgacatggag gcccagaata ccctccttga cagtcttgac
     3121 gtgcgcagct caggggcatg atgtgactgt cgcccgtaca tttagcccat acatccccat
     3181 gtataatcat ttgcatccat acattttgat ggccgcacgg cgcgaagcaa aaattacggc
     3241 tcctcgctgc agacctgcga gcagggaaac gctcccctca cagacgcgtt gaattgtccc
     3301 cacgccgcgc ccctgtagag aaatataaaa ggttaggatt tgccactgag gttcttcttt
     3361 catatacttc cttttaaaat cttgctagga tacagttctc acatcacatc cgaacataaa
     3421 caaccatggg taaaaagcct gaactcaccg cgacgtctgt cgagaagttt ctgatcgaaa
     3481 agttcgacag cgtctccgac ctgatgcagc tctcggaggg cgaagaatct cgtgctttca
     3541 gcttcgatgt aggagggcgt ggatatgtcc tgcgggtaaa tagctgcgcc gatggtttct
     3601 acaaagatcg ttatgtttat cggcactttg catcggccgc gctcccgatt ccggaagtgc
     3661 ttgacattgg ggaattcagc gagagcctga cctattgcat ctcccgccgt gcacagggtg
     3721 tcacgttgca agacctgcct gaaaccgaac tgcccgctgt tctgcagccg gtcgcggagg
     3781 ccatggatgc gatcgctgcg gccgatctta gccagacgag cgggttcggc ccattcggac
     3841 cgcaaggaat cggtcaatac actacatggc gtgatttcat atgcgcgatt gctgatcccc
     3901 atgtgtatca ctggcaaact gtgatggacg acaccgtcag tgcgtccgtc gcgcaggctc
     3961 tcgatgagct gatgctttgg gccgaggact gccccgaagt ccggcacctc gtgcacgcgg
     4021 atttcggctc caacaatgtc ctgacggaca atggccgcat aacagcggtc attgactgga
     4081 gcgaggcgat gttcggggat tcccaatacg aggtcgccaa catcttcttc tggaggccgt
     4141 ggttggcttg tatggagcag cagacgcgct acttcgagcg gaggcatccg gagcttgcag
     4201 gatcgccgcg gctccgggcg tatatgctcc gcattggtct tgaccaactc tatcagagct
     4261 tggttgacgg caatttcgat gatgca
//
