LOCUS       sequence-328795-        5966 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of gRNA targeting the MAT locus .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5966)
  TITLE     Burgess Lab Plasmids
REFERENCE   2  (bases 1 to 5966)
  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: pMEL15_MATz
FEATURES             Location/Qualifiers
     source          1..5966
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     132..151
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     385..402
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    519..540
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(555..560,561..578,579..585)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    593..609
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     598..620
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     617..633
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     617..633
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     652..672
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        654..672
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        690..958
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     misc_RNA        979..1053
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      1058..1077
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     terminator      1083..1330
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(1348..1367)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1349..1367)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1377..1394)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1377..1393)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1386..1408)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(1621..1640)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    complement(1867..1900)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     terminator      complement(2048..2235)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     2156..2180
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     CDS             complement(2243..2815)
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     promoter        complement(2816..3159)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     protein_bind    complement(3214..3247)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    3436..3483
                     /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)."
     rep_origin      3464..4446
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     promoter        4473..4577
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4578..4646,4647..5438)
                     /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     complement(4796..4815)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(5609..5966,1..231)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
       61 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
      121 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
      181 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
      241 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
      301 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
      361 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca
      421 atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg
      481 tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat
      541 taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc
      601 ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagcg cgcaattaac
      661 cctcactaaa gggaacaaaa gctggagctt ctttgaaaag ataatgtatg attatgcttt
      721 cactcatatt tatacagaaa cttgatgttt tctttcgagt atatacaagg tgattacatg
      781 tacgtttgaa gtacaactct agattttgta gtgccctctt gggctagcgg taaaggtgcg
      841 cattttttca caccctacaa tgttctgttc aaaagatttt ggtcaaacgc tgtagaagtg
      901 aaagttggtg cgcatgtttc ggcgttcgaa acttctccgc agtgaaagat aaatgatcca
      961 gtaaaatttt ataaacccgt tttagagcta gaaatagcaa gttaaaataa ggctagtccg
     1021 ttatcaactt gaaaaagtgg caccgagtcg gtggtgcttt ttttgttttt tatgtcttcg
     1081 agtcatgtaa ttagttatgt cacgcttacg ttcacgccct ccccccacat ccgctctaac
     1141 cgaaaaggaa ggagttagac aacctgaagt ctaggtccct atttattttt ttatagttat
     1201 gttagtatta agaacgttat ttatatttca aatttttctt ttttttctgt acagacgcgt
     1261 gtacgcatgt aacattatac tgaaaacctt gcttgagaag gttttgggac gctcgaaggc
     1321 tttaatttgc ggccggtacc caattcgccc tatagtgagt cgtattacgc gcgctcactg
     1381 gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt
     1441 gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct
     1501 tcccaacagt tgcgcagcct gaatggcgaa tggacgcgcc ctgtagcggc gcattaagcg
     1561 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     1621 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     1681 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     1741 aacttgatta gggtgatggt tcacgtaatg ggccgttgaa cattcttagg ctggtcgaat
     1801 catttagaca cgggcatcgt cctctcgaaa ggtggcatag gccactagtg gatctgatat
     1861 cacctaataa cttcgtatag catacattat acgaagttat attaagggtt ctcgagagct
     1921 cgattacaac aggtgttgtc ctctgaggac ataaaataca caccgagatt catcaactca
     1981 ttgctggagt tagcatatct acaattgggt gaaatgggga gcgatttgca ggcatttgct
     2041 cggcatgccg gtagaggtgt ggtcaataag agcgacctca tgctatacct gagaaagcaa
     2101 cctgacctac aggaaagagt tactcaagaa taagaatttt cgttttaaaa cctaagagtc
     2161 actttaaaat ttgtatacac ttattttttt tataacttat ttaataataa aaatcataaa
     2221 tcataagaaa ttcgcctcga gattaggggc agggcatgct catgtagagc gcctgctcgc
     2281 cgtccgaggc ggtgccgtcg tacagggcgg tgtccaggcc gcagagggtg aaccccatcc
     2341 gccggtacgc gtggatcgcc ggtgcgttga cgttggtgac ctccagccag aggtgcccgg
     2401 cgccccgctc gcgggcgaac tccgtcgcga gccccatcaa cgcgcgcccg accccgtgcc
     2461 cccggtgctc cggggcgacc tcgatgtcct cgacggtcag ccggcggttc cagccggagt
     2521 acgagacgac cacgaagccc gccaggtcgc cgtcgtcccc gtacgcgacg aacgtccggg
     2581 agtccgggtc gccgtcctcc ccgtcgtccg attcgtcgtc cgattcgtcg tcggggaaca
     2641 ccttggtcag gggcgggtcc accggcacct cccgcagggt gaagccgtcc ccggtggcgg
     2701 tgacgcggaa gacggtgtcg gtggtgaagg acccatccag tgcctcgatg gcctcggcgt
     2761 cccccgggac actggtgcgg taccggtaag ccgtgtcgtc aagagtggta cccatggttg
     2821 tttatgttcg gatgtgatgt gagaactgta tcctagcaag attttaaaag gaagtatatg
     2881 aaagaagaac ctcagtggca aatcctaacc ttttatattt ctctacaggg gcgcggcgtg
     2941 gggacaattc aacgcgtctg tgaggggagc gtttccctgc tcgcaggtct gcagcgagga
     3001 gccgtaattt ttgcttcgcg ccgtgcggcc atcaaaatgt atggatgcaa atgattatac
     3061 atggggatgt atgggctaaa tgtacgggcg acagtcacat catgcccctg agctgcgcac
     3121 gtcaagactg tcaaggaggg tattctgggc ctccatgtcg ctggccgggt gacccggcgg
     3181 ggacgaggca agctaaacag atctctagac ctaataactt cgtatagcat acattatacg
     3241 aagttatatt aagggttgtc gacctgcagc gtacgaaggt gcctattgat gatctggcgg
     3301 aatgtctgcc gtgccatagc catgccttca catatagtac aggcaacacg cagatatagg
     3361 tgcgacgtga acagtgagct gtatgtgcgc agctcgcgtt gcattttcgg aagcgctcgt
     3421 tttcggaaac gctttgaagt tcctattccg aagttcctat tctctagaaa gtataggaac
     3481 ttcagagcgc ttttgaaaac caaaagcgct ctgaagacgc actttcaaaa aaccaaaaac
     3541 gcaccggact gtaacgagct actaaaatat tgcgaatacc gcttccacaa acattgctca
     3601 aaagtatctc tttgctatat atctctgtgc tatatcccta tataacctac ccatccacct
     3661 ttcgctcctt gaacttgcat ctaaactcga cctctacatt ttttatgttt atctctagta
     3721 ttactcttta gacaaaaaaa ttgtagtaag aactattcat agagtgaatc gaaaacaata
     3781 cgaaaatgta aacatttcct atacgtagta tatagagaca aaatagaaga aaccgttcat
     3841 aattttctga ccaatgaaga atcatcaacg ctatcacttt ctgttcacaa agtatgcgca
     3901 atccacatcg gtatagaata taatcgggga tgcctttatc ttgaaaaaat gcacccgcag
     3961 cttcgctagt aatcagtaaa cgcgggaagt ggagtcaggc tttttttatg gaagagaaaa
     4021 tagacaccaa agtagccttc ttctaacctt aacggaccta cagtgcaaaa agttatcaag
     4081 agactgcatt atagagcgca caaaggagaa aaaaagtaat ctaagatgct ttgttagaaa
     4141 aatagcgctc tcgggatgca tttttgtaga acaaaaaaga agtatagatt ctttgttggt
     4201 aaaatagcgc tctcgcgttg catttctgtt ctgtaaaaat gcagctcaga ttctttgttt
     4261 gaaaaattag cgctctcgcg ttgcattttt gttttacaaa aatgaagcac agattcttcg
     4321 ttggtaaaat agcgctttcg cgttgcattt ctgttctgta aaaatgcagc tcagattctt
     4381 tgtttgaaaa attagcgctc tcgcgttgca tttttgttct acaaaatgaa gcacagatgc
     4441 ttcgttcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     4501 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     4561 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     4621 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     4681 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     4741 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     4801 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
     4861 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     4921 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     4981 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     5041 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     5101 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     5161 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     5221 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     5281 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     5341 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     5401 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     5461 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     5521 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     5581 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     5641 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     5701 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
     5761 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     5821 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     5881 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     5941 gcacacagcc cagcttggag cgaacg
//
