LOCUS       sequence-353606-        6420 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of Sapphire for auxotrophic selection in the absence of 
            uracil.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6420)
  TITLE     Ralser Yeast Expression plasmids for auxotrophic selection
REFERENCE   2  (bases 1 to 6420)
  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: pU-Sapphire
FEATURES             Location/Qualifiers
     source          1..6420
                     /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             477..1280
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     rep_origin      complement(1472..1927)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1609..1630)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1821..1840
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2053..2075
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2067..2084
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2068..2084
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2091..2110
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2091..2109
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        2205..2871
                     /gene="S. cerevisiae TDH3"
                     /label=GAP promoter
                     /note="promoter for glyceraldehyde-3-phosphate 
                     dehydrogenase; also known as the TDH3 promoter"
     primer_bind     2741..2764
                     /label=GPDpro-F
                     /note="S. cerevisiae GPD promoter, forward primer"
     CDS             2896..3612
                     /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      3633..3820
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(3688..3712)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     promoter        complement(3839..3857)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3878..3894)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3878..3894)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3891..3913)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3902..3918
                     /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(3926..3932,3933..3950,3951..3956))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3971..3992
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4109..4126)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4280..4868)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4360..4379)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5039..5830,5831..5899))
                     /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     5662..5681
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5900..6004)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    join(6034..6420,1..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 gattcggtaa tctccgaaca gaaggaagaa cgaaggaagg
      361 agcacagact tagattggta tatatacgca tatgtagtgt tgaagaaaca tgaaattgcc
      421 cagtattctt aacccaactg cacagaacaa aaacctgcag gaaacgaaga taaatcatgt
      481 cgaaagctac atataaggaa cgtgctgcta ctcatcctag tcctgttgct gccaagctat
      541 ttaatatcat gcacgaaaag caaacaaact tgtgtgcttc attggatgtt cgtaccacca
      601 aggaattact ggagttagtt gaagcattag gtcccaaaat ttgtttacta aaaacacatg
      661 tggatatctt gactgatttt tccatggagg gcacagttaa gccgctaaag gcattatccg
      721 ccaagtacaa ttttttactc ttcgaagaca gaaaatttgc tgacattggt aatacagtca
      781 aattgcagta ctctgcgggt gtatacagaa tagcagaatg ggcagacatt acgaatgcac
      841 acggtgtggt gggcccaggt attgttagcg gtttgaagca ggcggcagaa gaagtaacaa
      901 aggaacctag aggccttttg atgttagcag aattgtcatg caagggctcc ctatctactg
      961 gagaatatac taagggtact gttgacattg cgaagagcga caaagatttt gttatcggct
     1021 ttattgctca aagagacatg ggtggaagag atgaaggtta cgattggttg attatgacac
     1081 ccggtgtggg tttagatgac aagggagacg cattgggtca acagtataga accgtggatg
     1141 atgtggtctc tacaggatct gacattatta ttgttggaag aggactattt gcaaagggaa
     1201 gggatgctaa ggtagagggt gaacgttaca gaaaagcagg ctgggaagca tatttgagaa
     1261 gatgcggcca gcaaaactaa aaaactgtat tataagtaaa tgcatgtata ctaaactcac
     1321 aaattagagc ttcaatttaa ttatatcagt tattacccta tgcggtgtga aataccgcac
     1381 agatgcgtaa ggagaaaata ccgcatcagg cgcggatcct atgcggtgtg aaataccgca
     1441 cagatgcgta aggagaaaat accgcatcag gaaattgtaa gcgttaatat tttgttaaaa
     1501 ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa
     1561 atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac
     1621 aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag
     1681 ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt
     1741 aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg
     1801 gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca
     1861 agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag
     1921 ggcgcgtcca ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct
     1981 cttcgctatt acgccagctg gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa
     2041 cgccagggtt ttcccagtca cgacgttgta aaacgacggc cagtgaattg taatacgact
     2101 cactataggg cgaattggag ctccaccgcg gtggcggccg ctctagaact agtgggtccc
     2161 ccgggctgca ggaattcgat atcaagctta tcgataccgt cgactcatta tcaatactgc
     2221 catttcaaag aatacgtaaa taattaatag tagtgatttt cctaacttta tttagtcaaa
     2281 aaattagcct tttaattctg ctgtaacccg tacatgccca aaataggggg cgggttacac
     2341 agaatatata acatcgtagg tgtctgggtg aacagtttat tcctggcatc cactaaatat
     2401 aatggagccc gctttttaag ctggcatcca gaaaaaaaaa gaatcccagc accaaaatat
     2461 tgttttcttc accaaccatc agttcatagg tccattctct tagcgcaact acagagaaca
     2521 ggggcacaaa caggcaaaaa acgggcacaa cctcaatgga gtgatgcaac ctgcctggag
     2581 taaatgatga cacaaggcaa ttgacccacg catgtatcta tctcattttc ttacaccttc
     2641 tattaccttc tgctctctct gatttggaaa aagctgaaaa aaaaggttga aaccagttcc
     2701 ctgaaattat tcccctactt gactaataag tatataaaga cggtaggtat tgattgtaat
     2761 tctgtaaatc tatttcttaa acttcttaaa ttctactttt atagttagtc ttttttttag
     2821 ttttaaaaca ccaagaactt agtttcgaat aaacacacat aaacaaacaa aggtgacggt
     2881 gctggtttaa ttaacatgtc taaaggtgaa gaattattca ctggtgttgt cccaattttg
     2941 gttgaattag atggtgatgt taatggtcac aaattttctg tctccggtga aggtgaaggt
     3001 gatgctactt acggtaaatt gaccttaaaa tttatttgta ctactggtaa attgccagtt
     3061 ccatggccaa ccttagtcac tactttttct tatggtgttc aatgttttgc tagataccca
     3121 gatcatatga aacaacatga ctttttcaag tctgccatgc cagaaggtta tgttcaagaa
     3181 agaactattt ttttcaaaga tgacggtaac tacaagacca gagctgaagt caagtttgaa
     3241 ggtgatacct tagttaatag aatcgaatta aaaggtattg attttaaaga agatggtaac
     3301 attttaggtc acaaattgga atacaacttt aactctcaca atgtttacat catggctgac
     3361 aaacaaaaga atggtatcaa agttaacttc aaaattagac acaacattga agatggttct
     3421 gttcaattag ctgaccatta tcaacaaaat actccaattg gtgatggtcc agtcttgtta
     3481 ccagacaacc attacttatc cattcaatct gccttatcca aagatccaaa cgaaaagaga
     3541 gaccacatgg tcttgttaga atttgttact gctgctggta ttacccatgg tattgatgaa
     3601 ttgtacaaat aaggcgcgcc acttctaaat aagcgaattt cttatgattt atgattttta
     3661 ttattaaata agttataaaa aaaataagtg tatacaaatt ttaaagtgac tcttaggttt
     3721 taaaacgaaa attcttattc ttgagtaact ctttcctgta ggtcaggttg ctttctcagg
     3781 tatagtatga ggtcgctctt attgaccaca cctctaccgg cagtaccagc ttttgttccc
     3841 tttagtgagg gttaatttcg agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     3901 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     3961 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     4021 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     4081 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     4141 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     4201 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     4261 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     4321 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     4381 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     4441 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     4501 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     4561 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     4621 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     4681 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     4741 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     4801 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     4861 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     4921 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     4981 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     5041 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     5101 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     5161 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     5221 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     5281 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     5341 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     5401 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     5461 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     5521 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     5581 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     5641 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     5701 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     5761 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     5821 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     5881 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     5941 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     6001 cgcgcacatt tccccgaaaa gtgccacctg acggatcgct tgcctgtaac ttacacgcgc
     6061 ctcgtatctt ttaatgatgg aataatttgg gaatttactc tgtatttatt tatttttatg
     6121 ttttgtattt ggattttaga aagtaaataa agaaggtaga agagttacgg aatgaagaaa
     6181 aaaaaataaa caaaggttta aaaaatttca acaaaaagcg tactttacat atatatttat
     6241 tagacaagaa aagcagatta aatagatata cattcgatta acgataagta aaatgtaaaa
     6301 tcacaggatt ttcgtgtgtg gtcttctaca cagacaagat gaaacaattc ggcattaata
     6361 cctgagagca ggaagagcaa gataaaaggt agtatttgtt ggcgatcccc ctagagtctt
//
