LOCUS       sequence-350302-        6951 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of Sapphire for auxotrophic selection in the absence of 
            methionine.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6951)
  TITLE     Ralser Yeast Expression plasmids for auxotrophic selection
REFERENCE   2  (bases 1 to 6951)
  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: pM-Sapphire
FEATURES             Location/Qualifiers
     source          1..6951
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     165..183
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(221..243)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(376..1710)
                     /codon_start=1
                     /gene="S. cerevisiae MET17"
                     /gene_synonym="MET15; MET25"
                     /product="O-acetylserine and O-acetylhomoserine 
                     sulfhydrylase, required for sulfur amino acid synthesis"
                     /label=MET17
                     /translation="MPSHFDTVQLHAGQENPGDNAHRSRAVPIYATTSYVFENSKHGSQ
                     LFGLEVPGYVYSRFQNPTSNVLEERIAALEGGAAALAVSSGQAAQTLAIQGLAHTGDNI
                     VSTSYLYGGTYNQFKISFKRFGIEARFVEGDNPEEFEKVFDERTKAVYLETIGNPKYNV
                     PDFEKIVAIAHKHGIPVVVDNTFGAGGYFCQPIKYGADIVTHSATKWIGGHGTTIGGII
                     VDSGKFPWKDYPEKFPQFSQPAEGYHGTIYNEAYGNLAYIVHVRTELLRDLGPLMNPFA
                     SFLLLQGVETLSLRAERHGENALKLAKWLEQSPYVSWVSYPGLASHSHHENAKKYLSNG
                     FGGVLSFGVKDLPNADKETDPFKLSGAQVVDNLKLASNLANVGDAKTLVIAPYFTTHKQ
                     LNDKEKLASGVTKDLIRVSVGIEFIDDIIADFQQSFETVFAGQKP"
     rep_origin      complement(2003..2458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2140..2161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2352..2371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2584..2606
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2598..2615
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2599..2615
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2622..2641
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2622..2640
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        2736..3402
                     /gene="S. cerevisiae TDH3"
                     /label=GAP promoter
                     /note="promoter for glyceraldehyde-3-phosphate 
                     dehydrogenase; also known as the TDH3 promoter"
     primer_bind     3272..3295
                     /label=GPDpro-F
                     /note="S. cerevisiae GPD promoter, forward primer"
     CDS             3427..4143
                     /codon_start=1
                     /product="yeast-enhanced green fluorescent protein"
                     /label=yeGFP
                     /note="codons optimized for translation in S. cerevisiae 
                     and C. albicans"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFSYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSIQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGIDELYK"
     terminator      4164..4351
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(4219..4243)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     promoter        complement(4370..4388)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4409..4425)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4409..4425)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4422..4444)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4433..4449
                     /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)."
     promoter        complement(join(4457..4463,4464..4481,4482..4487))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4502..4523
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4640..4657)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4811..5399)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4891..4910)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5570..6361,6362..6430))
                     /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     6193..6212
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6431..6535)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    join(6565..6951,1..117)
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
ORIGIN
        1 ttacatcttc ggaaaacaaa aactattttt tctttaattt ctttttttac tttctatttt
       61 taatttatat atttatatta aaaaatttaa attataatta tttttatagc acgtgatgaa
      121 aaggacccta agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga
      181 ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc
      241 cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg
      301 cgtcagcggg tgttggcggt cgagcttgtg agagaaagta ggtttataca taattttaca
      361 actcattacg cacactcatg gtttttggcc agcgaaaaca gtttcaaaag attgctggaa
      421 gtctgcaata atgtcatcaa taaattcgat accaacagag acacgaatta agtccttggt
      481 aacaccagat gccaactttt ctttgtcatt taattgtttg tgggtagtga agtatggagc
      541 aatgactaag gtcttggcat caccaacatt ggccaagtta gaggcaagct ttaaattgtc
      601 aacaacttga gcaccagaaa gtttgaatgg gtcagtttcc ttgtcggcat ttggtaagtc
      661 ttttacaccg aaagataaga caccaccgaa accgttagat agatacttct tagcattttc
      721 atgatgagaa tgagatgcta aaccagggta tgaaacccaa gatacgtatg gggattgttc
      781 taaccatttg gctaacttca atgcattttc accgtgtctt tcagctctca aagataatgt
      841 ttcaacacct tgtagtagca agaaagaggc aaatgggttc atcaatggac ccaaatctct
      901 taatagttca gttctaacat gaacgatgta tgccaagtta ccgtaggctt cattgtagat
      961 agtaccgtga tatccttcgg caggttgaga gaattgaggg aacttttctg ggtagtcctt
     1021 ccatgggaac ttaccagagt caacaataat accaccgata gtagtaccat gaccaccaat
     1081 ccatttggta gcagaatgtg ttacaatatc agcaccgtat ttaattggct gacagaagta
     1141 accaccggca ccaaatgtgt tgtcaacgac aactggaata ccgtgtttgt gagcaattgc
     1201 aacaattttt tcaaaatccg gaacattgta ctttggatta ccaatggttt ccaaataaac
     1261 agccttggtt ctttcatcaa agaccttttc gaattcttct ggattgtcac cttcaacaaa
     1321 tctagcctcg ataccaaatc ttttgaacga gattttgaac tggttataag taccaccgta
     1381 taagtaagaa gtggaaacga tgttgtcacc agtgtgtgcc aaaccttgga tggcaagggt
     1441 ttgagcggct tgaccggagg aaacagccaa agcagcagca ccaccttcta aagcagcaat
     1501 tctttcttcc aaaacattac tggttgggtt ttggaaacgg gaatagacgt aacctggaac
     1561 ttctagacca aacaattgcg aaccatgctt agagttttcg aaaacataag aagtggtggc
     1621 gtaaattggt acagctctgg atctgtgagc attgtcacca gggttctctt ggccggcgtg
     1681 tagttgaaca gtatcgaaat gagatggcat tgtatggatg ggggtaatag aattgtatct
     1741 atgtatctga cgaccctgta ttacacgaag gaaatagtag atgaaaagac aagagagcaa
     1801 gaaaaaggaa aaacttcctt tctaacagac gctttactta accttatata tatcttattt
     1861 ttttcatcga gcgtgttcaa ttggacaagg tgccaccttt tcgacacttc ggttattatg
     1921 ttacacagtt ttcatgagga tggcggatcc tatgcggtgt gaaataccgc acagatgcgt
     1981 aaggagaaaa taccgcatca ggaaattgta agcgttaata ttttgttaaa attcgcgtta
     2041 aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat
     2101 aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca
     2161 ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc
     2221 ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta
     2281 aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg
     2341 gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg
     2401 gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc
     2461 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
     2521 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
     2581 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt gtaatacgac tcactatagg
     2641 gcgaattgga gctccaccgc ggtggcggcc gctctagaac tagtgggtcc cccgggctgc
     2701 aggaattcga tatcaagctt atcgataccg tcgactcatt atcaatactg ccatttcaaa
     2761 gaatacgtaa ataattaata gtagtgattt tcctaacttt atttagtcaa aaaattagcc
     2821 ttttaattct gctgtaaccc gtacatgccc aaaatagggg gcgggttaca cagaatatat
     2881 aacatcgtag gtgtctgggt gaacagttta ttcctggcat ccactaaata taatggagcc
     2941 cgctttttaa gctggcatcc agaaaaaaaa agaatcccag caccaaaata ttgttttctt
     3001 caccaaccat cagttcatag gtccattctc ttagcgcaac tacagagaac aggggcacaa
     3061 acaggcaaaa aacgggcaca acctcaatgg agtgatgcaa cctgcctgga gtaaatgatg
     3121 acacaaggca attgacccac gcatgtatct atctcatttt cttacacctt ctattacctt
     3181 ctgctctctc tgatttggaa aaagctgaaa aaaaaggttg aaaccagttc cctgaaatta
     3241 ttcccctact tgactaataa gtatataaag acggtaggta ttgattgtaa ttctgtaaat
     3301 ctatttctta aacttcttaa attctacttt tatagttagt ctttttttta gttttaaaac
     3361 accaagaact tagtttcgaa taaacacaca taaacaaaca aaggtgacgg tgctggttta
     3421 attaacatgt ctaaaggtga agaattattc actggtgttg tcccaatttt ggttgaatta
     3481 gatggtgatg ttaatggtca caaattttct gtctccggtg aaggtgaagg tgatgctact
     3541 tacggtaaat tgaccttaaa atttatttgt actactggta aattgccagt tccatggcca
     3601 accttagtca ctactttttc ttatggtgtt caatgttttg ctagataccc agatcatatg
     3661 aaacaacatg actttttcaa gtctgccatg ccagaaggtt atgttcaaga aagaactatt
     3721 tttttcaaag atgacggtaa ctacaagacc agagctgaag tcaagtttga aggtgatacc
     3781 ttagttaata gaatcgaatt aaaaggtatt gattttaaag aagatggtaa cattttaggt
     3841 cacaaattgg aatacaactt taactctcac aatgtttaca tcatggctga caaacaaaag
     3901 aatggtatca aagttaactt caaaattaga cacaacattg aagatggttc tgttcaatta
     3961 gctgaccatt atcaacaaaa tactccaatt ggtgatggtc cagtcttgtt accagacaac
     4021 cattacttat ccattcaatc tgccttatcc aaagatccaa acgaaaagag agaccacatg
     4081 gtcttgttag aatttgttac tgctgctggt attacccatg gtattgatga attgtacaaa
     4141 taaggcgcgc cacttctaaa taagcgaatt tcttatgatt tatgattttt attattaaat
     4201 aagttataaa aaaaataagt gtatacaaat tttaaagtga ctcttaggtt ttaaaacgaa
     4261 aattcttatt cttgagtaac tctttcctgt aggtcaggtt gctttctcag gtatagtatg
     4321 aggtcgctct tattgaccac acctctaccg gcagtaccag cttttgttcc ctttagtgag
     4381 ggttaatttc gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     4441 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     4501 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     4561 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     4621 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     4681 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     4741 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     4801 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     4861 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     4921 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     4981 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     5041 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     5101 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     5161 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     5221 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     5281 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     5341 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     5401 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     5461 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     5521 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     5581 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     5641 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     5701 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     5761 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     5821 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     5881 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     5941 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     6001 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     6061 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     6121 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     6181 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     6241 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     6301 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     6361 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     6421 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     6481 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     6541 ttccccgaaa agtgccacct gacggatcgc ttgcctgtaa cttacacgcg cctcgtatct
     6601 tttaatgatg gaataatttg ggaatttact ctgtatttat ttatttttat gttttgtatt
     6661 tggattttag aaagtaaata aagaaggtag aagagttacg gaatgaagaa aaaaaaataa
     6721 acaaaggttt aaaaaatttc aacaaaaagc gtactttaca tatatattta ttagacaaga
     6781 aaagcagatt aaatagatat acattcgatt aacgataagt aaaatgtaaa atcacaggat
     6841 tttcgtgtgt ggtcttctac acagacaaga tgaaacaatt cggcattaat acctgagagc
     6901 aggaagagca agataaaagg tagtatttgt tggcgatccc cctagagtct t
//
