LOCUS       sequence-350321-        6901 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of eCFP for auxotrophic selection in the absence of 
            methionine.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6901)
  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 6901)
  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: pM-eCFP
FEATURES             Location/Qualifiers
     source          1..6901
                     /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        2667..3333
                     /gene="S. cerevisiae TDH3"
                     /label=GAP promoter
                     /note="promoter for glyceraldehyde-3-phosphate 
                     dehydrogenase; also known as the TDH3 promoter"
     primer_bind     3203..3226
                     /label=GPDpro-F
                     /note="S. cerevisiae GPD promoter, forward primer"
     CDS             3358..4074
                     /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      4095..4282
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(4150..4174)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     promoter        complement(4320..4338)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4359..4375)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4359..4375)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4372..4394)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4383..4399
                     /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(4407..4413,4414..4431,4432..4437))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4452..4473
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4590..4607)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4761..5349)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4841..4860)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5520..6311,6312..6380))
                     /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     6143..6162
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6381..6485)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    join(6515..6901,1..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 ggtggctcat tatcaatact gccatttcaa agaatacgta
     2701 aataattaat agtagtgatt ttcctaactt tatttagtca aaaaattagc cttttaattc
     2761 tgctgtaacc cgtacatgcc caaaataggg ggcgggttac acagaatata taacatcgta
     2821 ggtgtctggg tgaacagttt attcctggca tccactaaat ataatggagc ccgcttttta
     2881 agctggcatc cagaaaaaaa aagaatccca gcaccaaaat attgttttct tcaccaacca
     2941 tcagttcata ggtccattct cttagcgcaa ctacagagaa caggggcaca aacaggcaaa
     3001 aaacgggcac aacctcaatg gagtgatgca acctgcctgg agtaaatgat gacacaaggc
     3061 aattgaccca cgcatgtatc tatctcattt tcttacacct tctattacct tctgctctct
     3121 ctgatttgga aaaagctgaa aaaaaaggtt gaaaccagtt ccctgaaatt attcccctac
     3181 ttgactaata agtatataaa gacggtaggt attgattgta attctgtaaa tctatttctt
     3241 aaacttctta aattctactt ttatagttag tctttttttt agttttaaaa caccaagaac
     3301 ttagtttcga ataaacacac ataaacaaac aaaggtgacg gtgctggttt aattaacatg
     3361 tctaaaggtg aagaattatt cactggtgtt gtcccaattt tggttgaatt agatggtgat
     3421 gttaatggtc acaaattttc tgtctccggt gaaggtgaag gtgatgctac ttacggtaaa
     3481 ttgaccttaa aatttatttg tactactggt aaattgccag ttccatggcc aaccttagtc
     3541 actactttaa cttggggtgt tcaatgtttt tctagatacc cagatcatat gaaacaacat
     3601 gactttttca agtctgccat gccagaaggt tatgttcaag aaagaactat ttttttcaaa
     3661 gatgacggta actacaagac cagagctgaa gtcaagtttg aaggtgatac cttagttaat
     3721 agaatcgaat taaaaggtat tgattttaaa gaagatggta acattttagg tcacaaattg
     3781 gaatacattt ataactctca caatgtttac atcactgctg acaaacaaaa gaatggtatc
     3841 aaagctaact tcaaaattag acacaacatt gaagatggtt ctgttcaatt agctgaccat
     3901 tatcaacaaa atactccaat tggtgatggt ccagtcttgt taccagacaa ccattactta
     3961 tccactcaat ctgccttatc caaagatcca aacgaaaaga gagaccacat ggtcttgtta
     4021 gaatttgtta ctgctgctgg tattacccat ggtattgatg aattgtacaa ataaggcgcg
     4081 ccacttctaa ataagcgaat ttcttatgat ttatgatttt tattattaaa taagttataa
     4141 aaaaaataag tgtatacaaa ttttaaagtg actcttaggt tttaaaacga aaattcttat
     4201 tcttgagtaa ctctttcctg taggtcaggt tgctttctca ggtatagtat gaggtcgctc
     4261 ttattgacca cacctctacc ggtcgacctg gagggggggc ccggtaccag cttttgttcc
     4321 ctttagtgag ggttaatttc gagcttggcg taatcatggt catagctgtt tcctgtgtga
     4381 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
     4441 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     4501 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     4561 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     4621 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     4681 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     4741 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     4801 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     4861 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     4921 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     4981 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     5041 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     5101 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     5161 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     5221 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     5281 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     5341 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     5401 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     5461 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     5521 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     5581 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     5641 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     5701 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     5761 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     5821 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     5881 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     5941 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     6001 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     6061 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     6121 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     6181 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     6241 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     6301 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     6361 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     6421 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     6481 ccgcgcacat ttccccgaaa agtgccacct gacggatcgc ttgcctgtaa cttacacgcg
     6541 cctcgtatct tttaatgatg gaataatttg ggaatttact ctgtatttat ttatttttat
     6601 gttttgtatt tggattttag aaagtaaata aagaaggtag aagagttacg gaatgaagaa
     6661 aaaaaaataa acaaaggttt aaaaaatttc aacaaaaagc gtactttaca tatatattta
     6721 ttagacaaga aaagcagatt aaatagatat acattcgatt aacgataagt aaaatgtaaa
     6781 atcacaggat tttcgtgtgt ggtcttctac acagacaaga tgaaacaatt cggcattaat
     6841 acctgagagc aggaagagca agataaaagg tagtatttgt tggcgatccc cctagagtct
     6901 t
//
