LOCUS       sequence-349775-        5727 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  ADH1t:T7p:cgRNA_HIS3_stop:tZ.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5727)
  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 5727)
  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: pEDJ508
FEATURES             Location/Qualifiers
     source          1..5727
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      62..227
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(117..141)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     269..288
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        269..287
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_RNA        1076..1150
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      1217..1263
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(1364..1382)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1378..1425
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        1456..1672
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             1673..2476
                     /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(2819..3407)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2899..2918)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3578..4369,4370..4438))
                     /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     4201..4220
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4439..4543)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4570..5552
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(5533..5580)
                     /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)."
ORIGIN
        1 acctgcactc aacggaatgc gtgcgatcgt tggtagatac gttgttgaca cttctaaata
       61 agcgaatttc ttatgattta tgatttttat tattaaataa gttataaaaa aaataagtgt
      121 atacaaattt taaagtgact cttaggtttt aaaacgaaaa ttcttattct tgagtaactc
      181 tttcctgtag gtcaggttgc tttctcaggt atagcatgag gtcgctcgtt aattaagagc
      241 tcgaatgcag ggaacaaaag ctggagctta atacgactca ctatagggag actacataag
      301 aacacctttg gtggagggaa catcgttggt accattgggc gaggtggctt ctcttatggc
      361 aaccgcaaga gccttgaacg cactctcact acggtgatga tcattcttgc ctcgcagaca
      421 atcaacgtgg agggtaattc tgctagcctc tgcaaagctt tcaagaaaat gcgggatcat
      481 ctcgcaagag agatctccta ctttctccct ttgcaaacca agttcgacaa ctgcgtacgg
      541 cctgttcgaa agatctacca ccgctctgga aagtgcctca tccaaaggcg caaatcctga
      601 tccaaacctt tttactccac gcacggcccc tagggcctct ttaaaagctt gaccgagagc
      661 aatcccgcag tcttcagtgg tgtgatggtc gtctatgtgt aagtcaccaa tgcactcaac
      721 gattagcgac cagccggaat gctagggtat gttaatatgg actaaaggag gcttttctgc
      781 aggtcgactc tagaggatcc ccgggtaccg agctcgaatt tttactaaca aatggtatta
      841 tttataacag ccagagcatg tatcatatgg tccagaaacc ctatacctgt gtggacgtta
      901 atcacttgcg attgtgtggc ctgttctgct actgcttctg cctctttttc tgggaagatc
      961 gagtgctcta tcgctagggg accacccttt aaagagatcg caatctgaat cttggtttca
     1021 tttgtaatac gctttactag ggctttctgc tctgttgtta gtaaaaattc ctcgagtttt
     1081 agagctagaa atagcaagtt aaaataaggc tagtccgtta tcaacttgaa aaagtggcac
     1141 cgagtcggtg gtgccctagc ataaccccgc ggggcctctt cgggggtctc gcggggtttt
     1201 ttgctgaaag aagcttcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgttt
     1261 tatctgttgt ttgtcgctgc ggccgcactc gagctcgacc tcgacctcca ctgagatccg
     1321 gctgctaaca aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta
     1381 gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa agatcgcgtg
     1441 cattccataa agcttttcaa ttcatctttt ttttttttgt tctttttttt gattccggtt
     1501 tctttgaaat ttttttgatt cggtaatctc cgaacagaag gaagaacgaa ggaaggagca
     1561 cagacttaga ttggtatata tacgcatatg tagtgttgaa gaaacatgaa attgcccagt
     1621 attcttaacc caactgcaca gaacaaaaac ctgcaggaaa cgaagataaa tcatgtcgaa
     1681 agctacatat aaggaacgtg ctgctactca tcctagtcct gttgctgcca agctatttaa
     1741 tatcatgcac gaaaagcaaa caaacttgtg tgcttcattg gatgttcgta ccaccaagga
     1801 attactggag ttagttgaag cattaggtcc caaaatttgt ttactaaaaa cacatgtgga
     1861 tatcttgact gatttttcca tggagggcac agttaagccg ctaaaggcat tatccgccaa
     1921 gtacaatttt ttactcttcg aagacagaaa atttgctgac attggtaata cagtcaaatt
     1981 gcagtactct gcgggtgtat acagaatagc agaatgggca gacattacga atgcacacgg
     2041 tgtggtgggc ccaggtattg ttagcggttt gaagcaggcg gcagaagaag taacaaagga
     2101 acctagaggc cttttgatgt tagcagaatt gtcatgcaag ggctccctat ctactggaga
     2161 atatactaag ggtactgttg acattgcgaa gagcgacaaa gattttgtta tcggctttat
     2221 tgctcaaaga gacatgggtg gaagagatga aggttacgat tggttgatta tgacacccgg
     2281 tgtgggttta gatgacaagg gagacgcatt gggtcaacag tatagaaccg tggatgatgt
     2341 ggtctctaca ggatctgaca ttattattgt tggaagagga ctatttgcaa agggaaggga
     2401 tgctaaggta gagggtgaac gttacagaaa agcaggctgg gaagcatatt tgagaagatg
     2461 cggccagcaa aactaaatct gtcatggaga ttgataagac ttttctagtt gcatatcttt
     2521 tatatttaaa tcttatctat tagttaattt tttgtaattt atccttatat atagtctggt
     2581 tattctaaaa tatcatttca gtatctaaaa attcccctct tttttcagtt atatcttaac
     2641 aggcgacagt ccaaatgttg atttatccca gtccgattca tcagggttgt gaagcatttt
     2701 gtcaatggtc gaaatcacat cagtaatagt gcctcttact tgcctcatag acatcgacga
     2761 gttgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     2821 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     2881 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     2941 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     3001 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     3061 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     3121 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     3181 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     3241 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     3301 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     3361 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     3421 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     3481 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     3541 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     3601 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     3661 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     3721 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     3781 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     3841 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     3901 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     3961 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     4021 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     4081 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     4141 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     4201 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     4261 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     4321 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     4381 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     4441 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     4501 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     4561 tgccacctga acgaagcatc tgtgcttcat tttgtagaac aaaaatgcaa cgcgagagcg
     4621 ctaatttttc aaacaaagaa tctgagctgc atttttacag aacagaaatg caacgcgaaa
     4681 gcgctatttt accaacgaag aatctgtgct tcatttttgt aaaacaaaaa tgcaacgcga
     4741 gagcgctaat ttttcaaaca aagaatctga gctgcatttt tacagaacag aaatgcaacg
     4801 cgagagcgct attttaccaa caaagaatct atacttcttt tttgttctac aaaaatgcat
     4861 cccgagagcg ctatttttct aacaaagcat cttagattac tttttttctc ctttgtgcgc
     4921 tctataatgc agtctcttga taactttttg cactgtaggt ccgttaaggt tagaagaagg
     4981 ctactttggt gtctattttc tcttccataa aaaaagcctg actccacttc ccgcgtttac
     5041 tgattactag cgaagctgcg ggtgcatttt ttcaagataa aggcatcccc gattatattc
     5101 tataccgatg tggattgcgc atactttgtg aacagaaagt gatagcgttg atgattcttc
     5161 attggtcaga aaattatgaa cggtttcttc tattttgtct ctatatacta cgtataggaa
     5221 atgtttacat tttcgtattg ttttcgattc actctatgaa tagttcttac tacaattttt
     5281 ttgtctaaag agtaatacta gagataaaca taaaaaatgt agaggtcgag tttagatgca
     5341 agttcaagga gcgaaaggtg gatgggtagg ttatataggg atatagcaca gagatatata
     5401 gcaaagagat acttttgagc aatgtttgtg gaagcggtat tcgcaatatt ttagtagctc
     5461 gttacagtcc ggtgcgtttt tggttttttg aaagtgcgtc ttcagagcgc ttttggtttt
     5521 caaaagcgct ctgaagttcc tatactttct agagaatagg aacttcggaa taggaacttc
     5581 aaagcgtttc cgaaaacgag cgcttccgaa aatgcaacgc gagctgcgca catacagctc
     5641 actgttcacg tcgcacctat atctgcgtgt tgcctgtata tatatataca tgagaagaac
     5701 ggcatagtgc gtgtttatgc ttaaatg
//
