LOCUS       sequence-349773-        6703 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Double gRNA for RNH1 RNH201 knock-out.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6703)
  AUTHORS   Jensen ED, Laloux M, Lehka BJ, Pedersen LE, Jakociunas T, Jensen MK,
            Keasling JD
  TITLE     A synthetic RNA-mediated evolution system in yeast.
  JOURNAL   Nucleic Acids Res. 2021 Sep 7;49(15):e88. doi: 10.1093/nar/gkab472.
  PUBMED    34107026
REFERENCE   2  (bases 1 to 6703)
  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"; journalName: "Nucleic 
            Acids Res."; date: "2021-09-7"; volume: "49(15):e88. doi"; pages: " 
            10.1093/nar/gkab472"
COMMENT     Sequence Label: pEDJ8
FEATURES             Location/Qualifiers
     source          1..6703
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        194..409
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             410..1213
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     rep_origin      complement(1344..1799)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1481..1502)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1693..1712
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(1878..2043)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     1964..1988
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     promoter        2220..2488
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     misc_RNA        2509..2583
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      2588..2607
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     promoter        2657..2925
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     misc_RNA        2946..3020
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      3025..3044
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     terminator      3110..3299
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(3371..3388)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3542..4130)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3622..3641)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4301..5092,5093..5161))
                     /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     4924..4943
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5162..5266)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5293..6635
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(6256..6303)
                     /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     6676..6694
                     /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 tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accataccac cttttcaatt catcattttt tttttattct tttttttgat ttcggtttcc
      241 ttgaaatttt tttgattcgg taatctccga acagaaggaa gaacgaagga aggagcacag
      301 acttagattg gtatatatac gcatatgtag tgttgaagaa acatgaaatt gcccagtatt
      361 cttaacccaa ctgcacagaa caaaaacctg caggaaacga agataaatca tgtcgaaagc
      421 tacatataag gaacgtgctg ctactcatcc tagtcctgtt gctgccaagc tatttaatat
      481 catgcacgaa aagcaaacaa acttgtgtgc ttcattggat gttcgtacca ccaaggaatt
      541 actggagtta gttgaagcat taggtcccaa aatttgttta ctaaaaacac atgtggatat
      601 cttgactgat ttttccatgg agggcacagt taagccgcta aaggcattat ccgccaagta
      661 caatttttta ctcttcgaag acagaaaatt tgctgacatt ggtaatacag tcaaattgca
      721 gtactctgcg ggtgtataca gaatagcaga atgggcagac attacgaatg cacacggtgt
      781 ggtgggccca ggtattgtta gcggtttgaa gcaggcggca gaagaagtaa caaaggaacc
      841 tagaggcctt ttgatgttag cagaattgtc atgcaagggc tccctatcta ctggagaata
      901 tactaagggt actgttgaca ttgcgaagag cgacaaagat tttgttatcg gctttattgc
      961 tcaaagagac atgggtggaa gagatgaagg ttacgattgg ttgattatga cacccggtgt
     1021 gggtttagat gacaagggag acgcattggg tcaacagtat agaaccgtgg atgatgtggt
     1081 ctctacagga tctgacatta ttattgttgg aagaggacta tttgcaaagg gaagggatgc
     1141 taaggtagag ggtgaacgtt acagaaaagc aggctgggaa gcatatttga gaagatgcgg
     1201 ccagcaaaac taaaaaactg tattataagt aaatgcatgt atactaaact cacaaattag
     1261 agcttcaatt taattatatc agttattacc ctatgcggtg tgaaataccg cacagatgcg
     1321 taaggagaaa ataccgcatc aggaaattgt aagcgttaat attttgttaa aattcgcgtt
     1381 aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta
     1441 taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc
     1501 actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg
     1561 cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact
     1621 aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt
     1681 ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc
     1741 ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc
     1801 cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta
     1861 ttacgccagc tgaattggag cgacctcatg ctatacctga gaaagcaacc tgacctacag
     1921 gaaagagtta ctcaagaata agaattttcg ttttaaaacc taagagtcac tttaaaattt
     1981 gtatacactt atttttttta taacttattt aataataaaa atcataaatc ataagaaatt
     2041 cgcttattta gaagtgtcaa caacgtatct accaacgatt tgaccctttt ccatcttttc
     2101 gtaaatttct ggcaaggtag acaagccgac aaccttgatt ggagacttga ccaaacctct
     2161 ggcgaagaat tgttaattaa gagctcgaat gcgtgcgata gggaacaaaa gctggagctt
     2221 ctttgaaaag ataatgtatg attatgcttt cactcatatt tatacagaaa cttgatgttt
     2281 tctttcgagt atatacaagg tgattacatg tacgtttgaa gtacaactct agattttgta
     2341 gtgccctctt gggctagcgg taaaggtgcg cattttttca caccctacaa tgttctgttc
     2401 aaaagatttt ggtcaaacgc tgtagaagtg aaagttggtg cgcatgtttc ggcgttcgaa
     2461 acttctccgc agtgaaagat aaatgatcgt ttgaagacca attagctggt tttagagcta
     2521 gaaatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg
     2581 gtggtgcttt ttttgttttt tatgtcttcg agtcatgtaa ttagttaagt gcaggtaggg
     2641 aacaaaagct ggagcttctt tgaaaagata atgtatgatt atgctttcac tcatatttat
     2701 acagaaactt gatgttttct ttcgagtata tacaaggtga ttacatgtac gtttgaagta
     2761 caactctaga ttttgtagtg ccctcttggg ctagcggtaa aggtgcgcat tttttcacac
     2821 cctacaatgt tctgttcaaa agattttggt caaacgctgt agaagtgaaa gttggtgcgc
     2881 atgtttcggc gttcgaaact tctccgcagt gaaagataaa tgatcgaagc tcagtcacgt
     2941 gtatggtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta tcaacttgaa
     3001 aaagtggcac cgagtcggtg gtgctttttt tgttttttat gtcttcgagt catgtaatta
     3061 gttaatcgcg tgcattcctc gagtaagctt ggtaccgcgg ctagctaaga tccgctctaa
     3121 ccgaaaagga aggagttaga caacctgaag tctaggtccc tatttatttt tttatagtta
     3181 tgttagtatt aagaacgtta tttatatttc aaatttttct tttttttctg tacagacgcg
     3241 tgtacgcatg taacattata ctgaaaacct tgcttgagaa ggttttggga cgctcgaaga
     3301 tccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     3361 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     3421 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     3481 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     3541 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     3601 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     3661 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     3721 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     3781 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     3841 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     3901 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     3961 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     4021 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     4081 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     4141 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     4201 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     4261 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     4321 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     4381 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     4441 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     4501 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     4561 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     4621 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     4681 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     4741 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     4801 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     4861 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     4921 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     4981 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     5041 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     5101 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     5161 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     5221 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     5281 aagtgccacc tgaacgaagc atctgtgctt cattttgtag aacaaaaatg caacgcgaga
     5341 gcgctaattt ttcaaacaaa gaatctgagc tgcattttta cagaacagaa atgcaacgcg
     5401 aaagcgctat tttaccaacg aagaatctgt gcttcatttt tgtaaaacaa aaatgcaacg
     5461 cgagagcgct aatttttcaa acaaagaatc tgagctgcat ttttacagaa cagaaatgca
     5521 acgcgagagc gctattttac caacaaagaa tctatacttc ttttttgttc tacaaaaatg
     5581 catcccgaga gcgctatttt tctaacaaag catcttagat tacttttttt ctcctttgtg
     5641 cgctctataa tgcagtctct tgataacttt ttgcactgta ggtccgttaa ggttagaaga
     5701 aggctacttt ggtgtctatt ttctcttcca taaaaaaagc ctgactccac ttcccgcgtt
     5761 tactgattac tagcgaagct gcgggtgcat tttttcaaga taaaggcatc cccgattata
     5821 ttctataccg atgtggattg cgcatacttt gtgaacagaa agtgatagcg ttgatgattc
     5881 ttcattggtc agaaaattat gaacggtttc ttctattttg tctctatata ctacgtatag
     5941 gaaatgttta cattttcgta ttgttttcga ttcactctat gaatagttct tactacaatt
     6001 tttttgtcta aagagtaata ctagagataa acataaaaaa tgtagaggtc gagtttagat
     6061 gcaagttcaa ggagcgaaag gtggatgggt aggttatata gggatatagc acagagatat
     6121 atagcaaaga gatacttttg agcaatgttt gtggaagcgg tattcgcaat attttagtag
     6181 ctcgttacag tccggtgcgt ttttggtttt ttgaaagtgc gtcttcagag cgcttttggt
     6241 tttcaaaagc gctctgaagt tcctatactt tctagagaat aggaacttcg gaataggaac
     6301 ttcaaagcgt ttccgaaaac gagcgcttcc gaaaatgcaa cgcgagctgc gcacatacag
     6361 ctcactgttc acgtcgcacc tatatctgcg tgttgcctgt atatatatat acatgagaag
     6421 aacggcatag tgcgtgttta tgcttaaatg cgtacttata tgcgtctatt tatgtaggat
     6481 gaaaggtagt ctagtacctc ctgtgatatt atcccattcc atgcggggta tcgtatgctt
     6541 ccttcagcac taccctttag ctgttctata tgctgccact cctcaattgg attagtctca
     6601 tccttcaatg ctatcatttc ctttgatatt ggatcatact aagaaaccat tattatcatg
     6661 acattaacct ataaaaatag gcgtatcacg aggccctttc gtc
//
