LOCUS       sequence-328822-        5657 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of dual gRNA targeting the MAT locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5657)
  TITLE     Burgess Lab Plasmids
REFERENCE   2  (bases 1 to 5657)
  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"
COMMENT     Sequence Label: pROS15_MATz
FEATURES             Location/Qualifiers
     source          1..5657
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(1..105)
                     /gene="bla"
                     /label=AmpR promoter
     protein_bind    205..238
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        293..636
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             637..1209
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     terminator      1217..1404
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(1272..1296)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     protein_bind    1552..1585
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        1708..1976
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     misc_RNA        1997..2071
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      2076..2095
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     rep_origin      2320..3301
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     terminator      complement(3347..3366)
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     misc_RNA        complement(3371..3445)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(3466..3734)
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     primer_bind     complement(3867..3884)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4038..4626)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4118..4137)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4797..5588,5589..5657))
                     /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     5420..5439
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
       61 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcactc gagactatat
      121 gtgaaggcat ggctatggca cggcagacat tccgccagat catcaatagg caccttcgta
      181 cgctgcaggt cgacaaccct taatataact tcgtataatg tatgctatac gaagttatta
      241 ggtctagaga tctgtttagc ttgcctcgtc cccgccgggt cacccggcca gcgacatgga
      301 ggcccagaat accctccttg acagtcttga cgtgcgcagc tcaggggcat gatgtgactg
      361 tcgcccgtac atttagccca tacatcccca tgtataatca tttgcatcca tacattttga
      421 tggccgcacg gcgcgaagca aaaattacgg ctcctcgctg cagacctgcg agcagggaaa
      481 cgctcccctc acagacgcgt tgaattgtcc ccacgccgcg cccctgtaga gaaatataaa
      541 aggttaggat ttgccactga ggttcttctt tcatatactt ccttttaaaa tcttgctagg
      601 atacagttct cacatcacat ccgaacataa acaaccatgg gtaccactct tgacgacacg
      661 gcttaccggt accgcaccag tgtcccgggg gacgccgagg ccatcgaggc actggatggg
      721 tccttcacca ccgacaccgt cttccgcgtc accgccaccg gggacggctt caccctgcgg
      781 gaggtgccgg tggacccgcc cctgaccaag gtgttccccg acgacgaatc ggacgacgaa
      841 tcggacgacg gggaggacgg cgacccggac tcccggacgt tcgtcgcgta cggggacgac
      901 ggcgacctgg cgggcttcgt ggtcgtctcg tactccggct ggaaccgccg gctgaccgtc
      961 gaggacatcg aggtcgcccc ggagcaccgg gggcacgggg tcgggcgcgc gttgatgggg
     1021 ctcgcgacgg agttcgcccg cgagcggggc gccgggcacc tctggctgga ggtcaccaac
     1081 gtcaacgcac cggcgatcca cgcgtaccgg cggatggggt tcaccctctg cggcctggac
     1141 accgccctgt acgacggcac cgcctcggac ggcgagcagg cgctctacat gagcatgccc
     1201 tgcccctaat ctcgaggcga atttcttatg atttatgatt tttattatta aataagttat
     1261 aaaaaaaata agtgtataca aattttaaag tgactcttag gttttaaaac gaaaattctt
     1321 attcttgagt aactctttcc tgtaggtcag gttgctttct caggtatagc atgaggtcgc
     1381 tcttattgac cacacctcta ccggcatgcc gagcaaatgc ctgcaaatcg ctccccattt
     1441 cacccaattg tagatatgct aactccagca atgagttgat gaatctcggt gtgtatttta
     1501 tgtcctcaga ggacaacacc tgttgtaatc gagctctcga gaacccttaa tataacttcg
     1561 tataatgtat gctatacgaa gttattaggt gatatcagat ccactagtgg cctatgccac
     1621 ctttcgagag gacgatgccc gtgtctaaat gattcgacca gcctaagaat gttcaacccc
     1681 tcactaaagg gaacaaaagc tggagcttct ttgaaaagat aatgtatgat tatgctttca
     1741 ctcatattta tacagaaact tgatgttttc tttcgagtat atacaaggtg attacatgta
     1801 cgtttgaagt acaactctag attttgtagt gccctcttgg gctagcggta aaggtgcgca
     1861 ttttttcaca ccctacaatg ttctgttcaa aagattttgg tcaaacgctg tagaagtgaa
     1921 agttggtgcg catgtttcgg cgttcgaaac ttctccgcag tgaaagataa atgatcactc
     1981 tacaaaacca aaaccagttt tagagctaga aatagcaagt taaaataagg ctagtccgtt
     2041 atcaacttga aaaagtggca ccgagtcggt ggtgcttttt ttgtttttta tgtcttcgag
     2101 tcatgtaatt agttatgtca cgcttacgtt cacgccctcc acgcatttaa gcataaacac
     2161 gcactatgcc gttcttctca tgtatatata tatacaggca acacgcagat ataggtgcga
     2221 cgtgaacagt gagctgtatg tgcgcagctc gcgttgcatt ttcggaagcg ctcgttttcg
     2281 gaaacgcttt gaagttccta ttccgaagtt cctattctgt agaaagtata ggaacctcag
     2341 agcgcttttg aaaaccaaaa gcgctctgaa gacgcacttt caaaaaacca aaaacgcacc
     2401 ggactgtaac gagctactaa aatattgcga ataccgcttc cacaaacatt gctcaaaagt
     2461 atctctttgc tatatatctc tgtgctatat ccctatataa cctacccatc cacctttcgc
     2521 tccttgaact tgcatctaaa ctcgacctct acatttttta tgtttatctc tagtattact
     2581 ctttagacaa aaaaattgta gtaagaacta ttcatagagt gaatcgaaaa caatacgaaa
     2641 atgtaaacat ttcctatatg tagtatatag agacaaaata gaagaaaccg ttcataattt
     2701 tctgaccaat gaagaatcat caacgctatc actttctgtt cacaaagtat gcgcaatcca
     2761 catcggtata gaatataatc ggggatgcct ttatcttgaa aaaatgcacc cgcagcttcg
     2821 ctagtaatca gtaaacgcgg gaagtggagt caggcttttt ttatggaaga gaaaatagac
     2881 accaaagtag ccttcttcta accttaacgg acctacagtg caaaaagtta tcaagagact
     2941 gcattataga gcgcacaaag gagaaaaaaa gtaatctaag atgctttgtt agaaaaatag
     3001 cgctctcggg atgcattttt gtagaacaaa aaagaagtat agattctttg ttggtaaaat
     3061 agcgctctcg cgttgcattt ctgttctgta aaaatgcagc tcagattctt tgtttgaaaa
     3121 attagcgctc tcgcgttgca tttttgtttt acaaaaatga agcacagatt cttcgttggt
     3181 aaaatagcgc tttcgcgttg catttctgtt ctgtaaaaat gcagctcaga ttctttgttt
     3241 gaaaaattag cgctctcgcg ttgcattttt gttctacaaa atgaagcaca gatgcttcgt
     3301 tggagggcgt gaacgtaagc gtgacataac taattacatg actcgaagac ataaaaaaca
     3361 aaaaaagcac caccgactcg gtgccacttt ttcaagttga taacggacta gccttatttt
     3421 aacttgctat ttctagctct aaaactcacc acgtacttca gcatagatca tttatctttc
     3481 actgcggaga agtttcgaac gccgaaacat gcgcaccaac tttcacttct acagcgtttg
     3541 accaaaatct tttgaacaga acattgtagg gtgtgaaaaa atgcgcacct ttaccgctag
     3601 cccaagaggg cactacaaaa tctagagttg tacttcaaac gtacatgtaa tcaccttgta
     3661 tatactcgaa agaaaacatc aagtttctgt ataaatatga gtgaaagcat aatcatacat
     3721 tatcttttca aagaagctcc agcttttgtt ccctttagtg agggtattca cgtagacgga
     3781 taggtatagc cagacatcag cagcatactt cgggaaccgt aggctctaga gagaggcggt
     3841 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     3901 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     3961 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     4021 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     4081 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     4141 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     4201 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     4261 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     4321 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     4381 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     4441 ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct
     4501 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     4561 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     4621 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     4681 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     4741 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     4801 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     4861 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     4921 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     4981 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     5041 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     5101 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     5161 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     5221 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     5281 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     5341 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     5401 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     5461 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     5521 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     5581 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     5641 aaatgttgaa tactcat
//
