LOCUS       sequence-353604-        7502 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of eCFP for auxotrophic selection in the absence of 
            leucine.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7502)
  AUTHORS   Yu JSL, Correia-Melo C, Zorrilla F, Herrera-Dominguez L, Wu MY, 
            Hartl J, Campbell K, Blasche S, Kreidl M, Egger AS, Messner CB, 
            Demichev V, Freiwald A, Mulleder M, Howell M, Berman J, Patil KR, 
            Alam MT, Ralser M
  TITLE     Microbial communities form rich extracellular metabolomes that 
            foster metabolic interactions and promote drug tolerance.
  JOURNAL   Nat Microbiol. 2022 Apr;7(4):542-555. doi: 
            10.1038/s41564-022-01072-5. Epub 2022 Mar 21.
  PUBMED    35314781
REFERENCE   2  (bases 1 to 7502)
  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"; doi: 
            "10.1038/s41564-022-01072-5"; journalName: "Nat Microbiol"; date: 
            "2022-04"; volume: "7"; issue: "4"; pages: "542-555"
COMMENT     Sequence Label: pL-eCFP
FEATURES             Location/Qualifiers
     source          1..7502
                     /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        3268..3934
                     /gene="S. cerevisiae TDH3"
                     /label=GAP promoter
                     /note="promoter for glyceraldehyde-3-phosphate 
                     dehydrogenase; also known as the TDH3 promoter"
     primer_bind     3804..3827
                     /label=GPDpro-F
                     /note="S. cerevisiae GPD promoter, forward primer"
     CDS             3959..4675
                     /codon_start=1
                     /product="yeast-enhanced green fluorescent protein"
                     /label=yeGFP
                     /note="codons optimized for translation in S. cerevisiae 
                     and C. albicans"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYIYNSHNVYITADKQKNGIKA
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGIDELYK"
     terminator      4696..4883
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(4751..4775)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     promoter        complement(4921..4939)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4960..4976)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4960..4976)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4973..4995)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4984..5000
                     /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(5008..5014,5015..5032,5033..5038))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5053..5074
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5191..5208)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5362..5950)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5442..5461)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6121..6912,6913..6981))
                     /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     6744..6763
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6982..7086)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    join(7116..7502,1..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 cggtggctca ttatcaatac tgccatttca aagaatacgt
     3301 aaataattaa tagtagtgat tttcctaact ttatttagtc aaaaaattag ccttttaatt
     3361 ctgctgtaac ccgtacatgc ccaaaatagg gggcgggtta cacagaatat ataacatcgt
     3421 aggtgtctgg gtgaacagtt tattcctggc atccactaaa tataatggag cccgcttttt
     3481 aagctggcat ccagaaaaaa aaagaatccc agcaccaaaa tattgttttc ttcaccaacc
     3541 atcagttcat aggtccattc tcttagcgca actacagaga acaggggcac aaacaggcaa
     3601 aaaacgggca caacctcaat ggagtgatgc aacctgcctg gagtaaatga tgacacaagg
     3661 caattgaccc acgcatgtat ctatctcatt ttcttacacc ttctattacc ttctgctctc
     3721 tctgatttgg aaaaagctga aaaaaaaggt tgaaaccagt tccctgaaat tattccccta
     3781 cttgactaat aagtatataa agacggtagg tattgattgt aattctgtaa atctatttct
     3841 taaacttctt aaattctact tttatagtta gtcttttttt tagttttaaa acaccaagaa
     3901 cttagtttcg aataaacaca cataaacaaa caaaggtgac ggtgctggtt taattaacat
     3961 gtctaaaggt gaagaattat tcactggtgt tgtcccaatt ttggttgaat tagatggtga
     4021 tgttaatggt cacaaatttt ctgtctccgg tgaaggtgaa ggtgatgcta cttacggtaa
     4081 attgacctta aaatttattt gtactactgg taaattgcca gttccatggc caaccttagt
     4141 cactacttta acttggggtg ttcaatgttt ttctagatac ccagatcata tgaaacaaca
     4201 tgactttttc aagtctgcca tgccagaagg ttatgttcaa gaaagaacta tttttttcaa
     4261 agatgacggt aactacaaga ccagagctga agtcaagttt gaaggtgata ccttagttaa
     4321 tagaatcgaa ttaaaaggta ttgattttaa agaagatggt aacattttag gtcacaaatt
     4381 ggaatacatt tataactctc acaatgttta catcactgct gacaaacaaa agaatggtat
     4441 caaagctaac ttcaaaatta gacacaacat tgaagatggt tctgttcaat tagctgacca
     4501 ttatcaacaa aatactccaa ttggtgatgg tccagtcttg ttaccagaca accattactt
     4561 atccactcaa tctgccttat ccaaagatcc aaacgaaaag agagaccaca tggtcttgtt
     4621 agaatttgtt actgctgctg gtattaccca tggtattgat gaattgtaca aataaggcgc
     4681 gccacttcta aataagcgaa tttcttatga tttatgattt ttattattaa ataagttata
     4741 aaaaaaataa gtgtatacaa attttaaagt gactcttagg ttttaaaacg aaaattctta
     4801 ttcttgagta actctttcct gtaggtcagg ttgctttctc aggtatagta tgaggtcgct
     4861 cttattgacc acacctctac cggtcgacct ggaggggggg cccggtacca gcttttgttc
     4921 cctttagtga gggttaattt cgagcttggc gtaatcatgg tcatagctgt ttcctgtgtg
     4981 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
     5041 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
     5101 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     5161 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     5221 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     5281 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     5341 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     5401 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     5461 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     5521 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     5581 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     5641 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     5701 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     5761 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     5821 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     5881 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     5941 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     6001 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     6061 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     6121 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     6181 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     6241 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     6301 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     6361 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     6421 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     6481 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     6541 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     6601 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     6661 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     6721 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     6781 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     6841 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     6901 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     6961 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     7021 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     7081 tccgcgcaca tttccccgaa aagtgccacc tgacggatcg cttgcctgta acttacacgc
     7141 gcctcgtatc ttttaatgat ggaataattt gggaatttac tctgtattta tttattttta
     7201 tgttttgtat ttggatttta gaaagtaaat aaagaaggta gaagagttac ggaatgaaga
     7261 aaaaaaaata aacaaaggtt taaaaaattt caacaaaaag cgtactttac atatatattt
     7321 attagacaag aaaagcagat taaatagata tacattcgat taacgataag taaaatgtaa
     7381 aatcacagga ttttcgtgtg tggtcttcta cacagacaag atgaaacaat tcggcattaa
     7441 tacctgagag caggaagagc aagataaaag gtagtatttg ttggcgatcc ccctagagtc
     7501 tt
//
