LOCUS       sequence-350974-        6566 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of Sapphire for auxotrophic selection in the absence of 
            lysine.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6566)
  TITLE     Ralser Yeast Expression plasmids for auxotrophic selection
REFERENCE   2  (bases 1 to 6566)
  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: pH-Sapphire
FEATURES             Location/Qualifiers
     source          1..6566
                     /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"
     primer_bind     343..362
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        509..696
                     /gene="S. cerevisiae HIS3"
                     /label=HIS3 promoter
     CDS             697..1359
                     /codon_start=1
                     /gene="S. cerevisiae HIS3"
                     /product="imidazoleglycerol-phosphate dehydratase, required
                     for histidine biosynthesis"
                     /label=HIS3
                     /note="yeast auxotrophic marker"
                     /translation="MTEQKALVKRITNETKIQIAISLKGGPLAIEHSIFPEKEAEAVAE
                     QATQSQVINVHTGIGFLDHMIHALAKHSGWSLIVECIGDLHIDDHHTTEDCGIALGQAF
                     KEALGAVRGVKRFGSGFAPLDEALSRAVVDLSNRPYAVVELGLQREKVGDLSCEMIPHF
                     LESFAEASRITLHVDCLRGKNDHHRSESAFKALAVAIREATSPNGTNDVPSTKGVLM"
     rep_origin      complement(1618..2073)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1755..1776)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1967..1986
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2199..2221
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2213..2230
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2214..2230
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2237..2256
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2237..2255
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        2351..3017
                     /gene="S. cerevisiae TDH3"
                     /label=GAP promoter
                     /note="promoter for glyceraldehyde-3-phosphate 
                     dehydrogenase; also known as the TDH3 promoter"
     primer_bind     2887..2910
                     /label=GPDpro-F
                     /note="S. cerevisiae GPD promoter, forward primer"
     CDS             3042..3758
                     /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      3779..3966
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(3834..3858)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     promoter        complement(3985..4003)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4024..4040)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4024..4040)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4037..4059)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4048..4064
                     /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(4072..4078,4079..4096,4097..4102))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4117..4138
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4255..4272)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4426..5014)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4506..4525)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5185..5976,5977..6045))
                     /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     5808..5827
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6046..6150)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    join(6180..6566,1..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 tgttggcgga cgtcggggct ggcttaacta tgcggcatca gagcagattg
      361 tactgagagt gcaccataaa ttcccgtttt aagagcttgg tgagcgctag gagtcactgc
      421 caggtatcgt ttgaacacgg cattagtcag ggaagtcata acacagtcct ttcccgcaat
      481 tttctttttc tattactctt ggcctcctct agtacactct atattttttt atgcctcggt
      541 aatgattttc attttttttt ttccacctag cggatgactc tttttttttc ttagcgattg
      601 gcattatcac ataatgaatt atacattata taaagtaatg tgatttcttc gaagaatata
      661 ctaaaaaatg agcaggcaag ataaacgaag gcaaagatga cagagcagaa agccctagta
      721 aagcgtatta caaatgaaac caagattcag attgcgatct ctttaaaggg tggtccccta
      781 gcgatagagc actcgatctt cccagaaaaa gaggcagaag cagtagcaga acaggccaca
      841 caatcgcaag tgattaacgt ccacacaggt atagggtttc tggaccatat gatacatgct
      901 ctggccaagc attccggctg gtcgctaatc gttgagtgca ttggtgactt acacatagac
      961 gaccatcaca ccactgaaga ctgcgggatt gctctcggtc aagcttttaa agaggcccta
     1021 ggggccgtgc gtggagtaaa aaggtttgga tcaggatttg cgcctttgga tgaggcactt
     1081 tccagagcgg tggtagatct ttcgaacagg ccgtacgcag ttgtcgaact tggtttgcaa
     1141 agggagaaag taggagatct ctcttgcgag atgatcccgc attttcttga aagctttgca
     1201 gaggctagca gaattaccct ccacgttgat tgtctgcgag gcaagaatga tcatcaccgt
     1261 agtgagagtg cgttcaaggc tcttgcggtt gccataagag aagccacctc gcccaatggt
     1321 accaacgatg ttccctccac caaaggtgtt cttatgtagt gacaccgatt atttaaagct
     1381 gcagcatacg atatatatac atgtgtatat atgtatacct atgaatgtca gtaagtatgt
     1441 atacgaacag tatgatactg aagatgacaa ggtaatgcat cattctatac gtgtcattct
     1501 gaacgaggcg cgctttcctt ttttcttttt gctttttctt tttttttctc ttgaactcga
     1561 gatcctatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggaaa
     1621 ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt
     1681 ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag
     1741 ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg
     1801 tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat
     1861 caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc
     1921 gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga
     1981 aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac
     2041 ccgccgcgct taatgcgccg ctacagggcg cgtccattcg ccattcaggc tgcgcaactg
     2101 ttgggaaggg cgatcggtgc gggcctcttt gctattacgc cagctggcga aagggggatg
     2161 tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac gttgtaaaac
     2221 gacggccagt gaattgtaat acgactcact atagggcgaa ttggagctcc accgcggtgg
     2281 cggccgctct agaactagtg ggtcccccgg gctgcaggaa ttcgatatca agcttatcga
     2341 taccgtcgac tcattatcaa tactgccatt tcaaagaata cgtaaataat taatagtagt
     2401 gattttccta actttattta gtcaaaaaat tagcctttta attctgctgt aacccgtaca
     2461 tgcccaaaat agggggcggg ttacacagaa tatataacat cgtaggtgtc tgggtgaaca
     2521 gtttattcct ggcatccact aaatataatg gagcccgctt tttaagctgg catccagaaa
     2581 aaaaaagaat cccagcacca aaatattgtt ttcttcacca accatcagtt cataggtcca
     2641 ttctcttagc gcaactacag agaacagggg cacaaacagg caaaaaacgg gcacaacctc
     2701 aatggagtga tgcaacctgc ctggagtaaa tgatgacaca aggcaattga cccacgcatg
     2761 tatctatctc attttcttac accttctatt accttctgct ctctctgatt tggaaaaagc
     2821 tgaaaaaaaa ggttgaaacc agttccctga aattattccc ctacttgact aataagtata
     2881 taaagacggt aggtattgat tgtaattctg taaatctatt tcttaaactt cttaaattct
     2941 acttttatag ttagtctttt ttttagtttt aaaacaccaa gaacttagtt tcgaataaac
     3001 acacataaac aaacaaaggt gacggtgctg gtttaattaa catgtctaaa ggtgaagaat
     3061 tattcactgg tgttgtccca attttggttg aattagatgg tgatgttaat ggtcacaaat
     3121 tttctgtctc cggtgaaggt gaaggtgatg ctacttacgg taaattgacc ttaaaattta
     3181 tttgtactac tggtaaattg ccagttccat ggccaacctt agtcactact ttttcttatg
     3241 gtgttcaatg ttttgctaga tacccagatc atatgaaaca acatgacttt ttcaagtctg
     3301 ccatgccaga aggttatgtt caagaaagaa ctattttttt caaagatgac ggtaactaca
     3361 agaccagagc tgaagtcaag tttgaaggtg ataccttagt taatagaatc gaattaaaag
     3421 gtattgattt taaagaagat ggtaacattt taggtcacaa attggaatac aactttaact
     3481 ctcacaatgt ttacatcatg gctgacaaac aaaagaatgg tatcaaagtt aacttcaaaa
     3541 ttagacacaa cattgaagat ggttctgttc aattagctga ccattatcaa caaaatactc
     3601 caattggtga tggtccagtc ttgttaccag acaaccatta cttatccatt caatctgcct
     3661 tatccaaaga tccaaacgaa aagagagacc acatggtctt gttagaattt gttactgctg
     3721 ctggtattac ccatggtatt gatgaattgt acaaataagg cgcgccactt ctaaataagc
     3781 gaatttctta tgatttatga tttttattat taaataagtt ataaaaaaaa taagtgtata
     3841 caaattttaa agtgactctt aggttttaaa acgaaaattc ttattcttga gtaactcttt
     3901 cctgtaggtc aggttgcttt ctcaggtata gtatgaggtc gctcttattg accacacctc
     3961 taccggcagt accagctttt gttcccttta gtgagggtta atttcgagct tggcgtaatc
     4021 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     4081 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     4141 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     4201 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     4261 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     4321 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     4381 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     4441 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     4501 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     4561 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     4621 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     4681 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     4741 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     4801 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     4861 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     4921 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     4981 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     5041 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     5101 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     5161 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     5221 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     5281 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     5341 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     5401 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     5461 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     5521 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     5581 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     5641 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     5701 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     5761 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     5821 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     5881 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     5941 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     6001 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     6061 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     6121 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgg
     6181 atcgcttgcc tgtaacttac acgcgcctcg tatcttttaa tgatggaata atttgggaat
     6241 ttactctgta tttatttatt tttatgtttt gtatttggat tttagaaagt aaataaagaa
     6301 ggtagaagag ttacggaatg aagaaaaaaa aataaacaaa ggtttaaaaa atttcaacaa
     6361 aaagcgtact ttacatatat atttattaga caagaaaagc agattaaata gatatacatt
     6421 cgattaacga taagtaaaat gtaaaatcac aggattttcg tgtgtggtct tctacacaga
     6481 caagatgaaa caattcggca ttaatacctg agagcaggaa gagcaagata aaaggtagta
     6541 tttgttggcg atccccctag agtctt
//
