LOCUS       sequence-350303-        7552 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of Sapphire for auxotrophic selection in the absence of 
            leucine.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7552)
  TITLE     Ralser Yeast Expression plasmids for auxotrophic selection
REFERENCE   2  (bases 1 to 7552)
  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: pL-Sapphire
FEATURES             Location/Qualifiers
     source          1..7552
                     /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"
     promoter        567..974
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             975..2069
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKADAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     rep_origin      complement(2604..3059)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2741..2762)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2953..2972
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3185..3207
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3199..3216
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3200..3216
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3223..3242
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3223..3241
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        3337..4003
                     /gene="S. cerevisiae TDH3"
                     /label=GAP promoter
                     /note="promoter for glyceraldehyde-3-phosphate 
                     dehydrogenase; also known as the TDH3 promoter"
     primer_bind     3873..3896
                     /label=GPDpro-F
                     /note="S. cerevisiae GPD promoter, forward primer"
     CDS             4028..4744
                     /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      4765..4952
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(4820..4844)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     promoter        complement(4971..4989)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5010..5026)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5010..5026)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5023..5045)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5034..5050
                     /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(5058..5064,5065..5082,5083..5088))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5103..5124
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5241..5258)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5412..6000)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5492..5511)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6171..6962,6963..7031))
                     /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     6794..6813
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7032..7136)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    join(7166..7552,1..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 tgttggcggc gcgccatcga ggagaacttc tagtatatct acatacctaa
      361 tattattgcc ttattaaaaa tggaatccca acaattacat caaaatccac attctcttca
      421 aaatcaattg tcctgtactt ccttgttcat gtgtgttcaa aaacgttata tttataggat
      481 aattatactc tatttctcaa caagtaattg gttgtttggc cgagcggtct aaggcgcctg
      541 attcaagaaa tatcttgacc gcagttaact gtgggaatac tcaggtatcg taagatgcaa
      601 gagttcgaat ctcttagcaa ccattatttt tttcctcaac ataacgagaa cacacagggg
      661 cgctatcgca cagaatcaaa ttcgatgact ggaaattttt tgttaatttc agaggtcgcc
      721 tgacgcatat acctttttca actgaaaaat tgggagaaaa aggaaaggtg agagcgccgg
      781 aaccggcttt tcatatagaa tagagaagcg ttcatgacta aatgcttgca tcacaatact
      841 tgaagttgac aatattattt aaggacctat tgttttttcc aataggtggt tagcaatcgt
      901 cttactttct aacttttctt accttttaca tttcagcaat atatatatat atatttcaag
      961 gatataccat tctaatgtct gcccctaaga agatcgtcgt tttgccaggt gaccacgttg
     1021 gtcaagaaat cacagccgaa gccattaagg ttcttaaagc tatttctgat gttcgttcca
     1081 atgtcaagtt cgatttcgaa aatcatttaa ttggtggtgc tgctatcgat gctacaggtg
     1141 ttccacttcc agatgaggcg ctggaagcct ccaagaaggc tgatgccgtt ttgttaggtg
     1201 ctgtgggtgg tcctaaatgg ggtaccggta gtgttagacc tgaacaaggt ttactaaaaa
     1261 tccgtaaaga acttcaattg tacgccaact taagaccatg taactttgca tccgactctc
     1321 ttttagactt atctccaatc aagccacaat ttgctaaagg tactgacttc gttgttgtca
     1381 gagaattagt gggaggtatt tactttggta agagaaagga agacgatggt gatggtgtcg
     1441 cttgggatag tgaacaatac accgttccag aagtgcaaag aatcacaaga atggccgctt
     1501 tcatggccct acaacatgag ccaccattgc ctatttggtc cttggataaa gctaatgttt
     1561 tggcctcttc aagattatgg agaaaaactg tggaggaaac catcaagaac gaattcccta
     1621 cattgaaggt tcaacatcaa ttgattgatt ctgccgccat gatcctagtt aagaacccaa
     1681 cccacctaaa tggtattata atcaccagca acatgtttgg tgatatcatc tccgatgaag
     1741 cctccgttat cccaggttcc ttgggtttgt tgccatctgc gtccttggcc tctttgccag
     1801 acaagaacac cgcatttggt ttgtacgaac catgccacgg ttctgctcca gatttgccaa
     1861 agaataaggt caaccctatc gccactatct tgtctgctgc aatgatgttg aaattgtcat
     1921 tgaacttgcc tgaagaaggt aaggccattg aagatgcagt taaaaaggtt ttggatgcag
     1981 gtatcagaac tggtgattta ggtggttcca acagtaccac cgaagtcggt gatgctgtcg
     2041 ccgaagaagt taagaaaatc cttgcttaaa aagattctct ttttttatga tatttgtaca
     2101 taaactttat aaatgaaatt cataatagaa acgacacgaa attacaaaat ggaatatgtt
     2161 catagggtag acgaaactat atacgcaatc tacatacatt tatcaagaag gagaaaaagg
     2221 aggatgtaaa ggaatacagg taagcaaatt gatactaatg gctcaacgtg ataaggaaaa
     2281 agaattgcac tttaacatta atattgacaa ggaggagggc accacacaaa aagttaggtg
     2341 taacagaaaa tcatgaaact atgattccta atttatatat tggaggattt tctctaaaaa
     2401 aaaaaaaata caacaaataa aaaacactca atgacctgac catttgatgg agtttaagtc
     2461 aataccttct tgaaccattt cccataatgg tgaaagttcc ctcaagaatt ttactctgtc
     2521 agaaacggcc ttacgacgta gtcgaggatc ctatgcggtg tgaaataccg cacagatgcg
     2581 taaggagaaa ataccgcatc aggaaattgt aagcgttaat attttgttaa aattcgcgtt
     2641 aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta
     2701 taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc
     2761 actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg
     2821 cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact
     2881 aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt
     2941 ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc
     3001 ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc
     3061 cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta
     3121 ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg
     3181 ttttcccagt cacgacgttg taaaacgacg gccagtgaat tgtaatacga ctcactatag
     3241 ggcgaattgg agctccaccg cggtggcggc cgctctagaa ctagtgggtc ccccgggctg
     3301 caggaattcg atatcaagct tatcgatacc gtcgactcat tatcaatact gccatttcaa
     3361 agaatacgta aataattaat agtagtgatt ttcctaactt tatttagtca aaaaattagc
     3421 cttttaattc tgctgtaacc cgtacatgcc caaaataggg ggcgggttac acagaatata
     3481 taacatcgta ggtgtctggg tgaacagttt attcctggca tccactaaat ataatggagc
     3541 ccgcttttta agctggcatc cagaaaaaaa aagaatccca gcaccaaaat attgttttct
     3601 tcaccaacca tcagttcata ggtccattct cttagcgcaa ctacagagaa caggggcaca
     3661 aacaggcaaa aaacgggcac aacctcaatg gagtgatgca acctgcctgg agtaaatgat
     3721 gacacaaggc aattgaccca cgcatgtatc tatctcattt tcttacacct tctattacct
     3781 tctgctctct ctgatttgga aaaagctgaa aaaaaaggtt gaaaccagtt ccctgaaatt
     3841 attcccctac ttgactaata agtatataaa gacggtaggt attgattgta attctgtaaa
     3901 tctatttctt aaacttctta aattctactt ttatagttag tctttttttt agttttaaaa
     3961 caccaagaac ttagtttcga ataaacacac ataaacaaac aaaggtgacg gtgctggttt
     4021 aattaacatg tctaaaggtg aagaattatt cactggtgtt gtcccaattt tggttgaatt
     4081 agatggtgat gttaatggtc acaaattttc tgtctccggt gaaggtgaag gtgatgctac
     4141 ttacggtaaa ttgaccttaa aatttatttg tactactggt aaattgccag ttccatggcc
     4201 aaccttagtc actacttttt cttatggtgt tcaatgtttt gctagatacc cagatcatat
     4261 gaaacaacat gactttttca agtctgccat gccagaaggt tatgttcaag aaagaactat
     4321 ttttttcaaa gatgacggta actacaagac cagagctgaa gtcaagtttg aaggtgatac
     4381 cttagttaat agaatcgaat taaaaggtat tgattttaaa gaagatggta acattttagg
     4441 tcacaaattg gaatacaact ttaactctca caatgtttac atcatggctg acaaacaaaa
     4501 gaatggtatc aaagttaact tcaaaattag acacaacatt gaagatggtt ctgttcaatt
     4561 agctgaccat tatcaacaaa atactccaat tggtgatggt ccagtcttgt taccagacaa
     4621 ccattactta tccattcaat ctgccttatc caaagatcca aacgaaaaga gagaccacat
     4681 ggtcttgtta gaatttgtta ctgctgctgg tattacccat ggtattgatg aattgtacaa
     4741 ataaggcgcg ccacttctaa ataagcgaat ttcttatgat ttatgatttt tattattaaa
     4801 taagttataa aaaaaataag tgtatacaaa ttttaaagtg actcttaggt tttaaaacga
     4861 aaattcttat tcttgagtaa ctctttcctg taggtcaggt tgctttctca ggtatagtat
     4921 gaggtcgctc ttattgacca cacctctacc ggcagtacca gcttttgttc cctttagtga
     4981 gggttaattt cgagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
     5041 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
     5101 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     5161 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     5221 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     5281 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     5341 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     5401 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     5461 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     5521 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     5581 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     5641 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     5701 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     5761 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     5821 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     5881 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     5941 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     6001 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     6061 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     6121 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     6181 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     6241 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     6301 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     6361 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     6421 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     6481 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     6541 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     6601 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     6661 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     6721 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     6781 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     6841 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     6901 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     6961 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     7021 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     7081 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     7141 tttccccgaa aagtgccacc tgacggatcg cttgcctgta acttacacgc gcctcgtatc
     7201 ttttaatgat ggaataattt gggaatttac tctgtattta tttattttta tgttttgtat
     7261 ttggatttta gaaagtaaat aaagaaggta gaagagttac ggaatgaaga aaaaaaaata
     7321 aacaaaggtt taaaaaattt caacaaaaag cgtactttac atatatattt attagacaag
     7381 aaaagcagat taaatagata tacattcgat taacgataag taaaatgtaa aatcacagga
     7441 ttttcgtgtg tggtcttcta cacagacaag atgaaacaat tcggcattaa tacctgagag
     7501 caggaagagc aagataaaag gtagtatttg ttggcgatcc ccctagagtc tt
//
