LOCUS       sequence-368107-        5830 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Testing the TEF1 promoter inserted with 4 TetO elements using a 
            green fluorescence protein as the reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5830)
  AUTHORS   Peng B, Bandari NC, Lu Z, Howard CB, Scott C, Trau M, Dumsday G, 
            Vickers CE
  TITLE     Engineering eukaryote-like regulatory circuits to expand artificial 
            control mechanisms for metabolic engineering in Saccharomyces 
            cerevisiae.
  JOURNAL   Commun Biol. 2022 Feb 16;5(1):135. doi: 10.1038/s42003-022-03070-z.
  PUBMED    35173283
REFERENCE   2  (bases 1 to 5830)
  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"; journalName: "Commun 
            Biol."; date: "2022-02-16"; volume: "5(1):135. doi"; pages: " 
            10.1038/s42003-022-03070-z"
COMMENT     Sequence Label: pILGFP9E3
FEATURES             Location/Qualifiers
     source          1..5830
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    433..466
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(1580..1597)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1580..1596)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1589..1611)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(1805..1824)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1924..1946
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1984..2002)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2070..2174
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2175..2243,2244..3035)
                     /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     complement(2393..2412)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3206..3794
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3695..3714
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3948..3965
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4082..4103
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4118..4123,4124..4141,4142..4148)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4156..4172
                     /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)."
     primer_bind     4161..4183
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4180..4196
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4180..4196
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     terminator      complement(4461..4731)
                     /gene="S. cerevisae PGK1"
                     /label=PGK1 terminator
                     /note="transcription terminator for 3-phosphoglycerate 
                     kinase gene"
     CDS             complement(4741..5454)
                     /codon_start=1
                     /product="yeast-enhanced green fluorescent protein"
                     /label=yeGFP
                     /note="codons optimized for translation in S. cerevisiae 
                     and C. albicans"
                     /translation="MSKGEELFTGIVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     protein_bind    5673..5691
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O1 for the tetR and tetA genes"
     protein_bind    5699..5717
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5733..5751
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5760..5778
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O1 for the tetR and tetA genes"
ORIGIN
        1 tttcgacgaa gaaaaagaaa cgaggcggtc tcttttttct tttccaaacc tttagtacgg
       61 gtaattaacg acaccctaga ggaagaaaga ggggaaattt agtatgctgt gcttgggtgt
      121 tttgaagtgg tacggcgatg cgcggagtcc gagaaaatct ggaagagtaa aaaaggagta
      181 gaaacatttt gaagctatgg tgtgtggggg atcacttgtg ggggattggg tgtgatgtaa
      241 ggattcgcgg tcctcgaaaa ttaaaagtcc aacgcgcctg ttgcttccta tgtgatatgt
      301 attatatgta atatgcataa atatatctac tgcattgtat tttgaacgta caaagtatgc
      361 attgtttata cgctattatc agccaaagtt gggtggtcgc tttctggagc tctttctcga
      421 gcaaccctta atataacttc gtataatgta tgctatacga agttattagg tctagagatc
      481 ccaatacaac agatcacgtg atctttttgt aagatgaagt tgaagtagtg ttgcaccgtg
      541 ccaatgcagg tggctattag attaaatatg tgatttgttc tattaagttt cctgtataat
      601 taatggggag cgctgattct cttttggtac gcttcccatc cagcatttct gtatctttca
      661 ccttcaacct taggatctct acccttggcg aaaagtcctc tgccaacaat gatgatatct
      721 gatccaccac ttacaacttc gtcgacggtt ctgtactgct gacccaatgc atcgcctttg
      781 tcgtctaaac ctacacctgg ggtcatgatt agccaatcaa acccttcttc tcttcctccc
      841 atatcgttct gagcaatgaa cccaataacg aaatctttat cactctttgc aatatcaacg
      901 gtacccttag tatattcacc gtgtgctaga gaacccttgg aagacaattc agcaagcatc
      961 aataatcccc ttggttcttt ggtgacctct tgcgcacctt gtttcaagcc agcaacaata
     1021 ccagcaccag taaccccgtg ggcgttggtg atatcagacc attctgcgat acggtaaacg
     1081 cccgatgtat attgtaattt gactgtgtta ccgatatcgg cgaattttct gtcctcaaat
     1141 atcaagaact tgtatttctc tgccaatgct ttcaatggaa cgacagtacc ctcataactg
     1201 aaatcatcca agatatcaac gtgtgttttc aaaaggcaaa tgtatggacc caacgtttca
     1261 acaagtttca atagctcatc agtcgaacga acgtcaagag aagcacacaa attggtcttc
     1321 ttttcatcca ttaaacgtaa aagtttcgat gcaaccggac ttgcatgagt ctcagctcta
     1381 ctggtatatg attttgtgga catgttttat cttcgtttcc tgcaggtttt tgttctgtgc
     1441 agttgggtta agaatactgg gcaatttcat gtttcttcaa cactacatat gcgtatatat
     1501 accaatctaa gtctgtgctc cttccttcgt tcttccttct gttcggagat taccgaatca
     1561 aaaaaattta aatgaattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg
     1621 gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg
     1681 aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggcgcc
     1741 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata tggtgcactc
     1801 tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg
     1861 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     1921 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa
     1981 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga
     2041 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     2101 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     2161 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     2221 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     2281 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     2341 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     2401 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     2461 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     2521 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     2581 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     2641 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     2701 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     2761 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     2821 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     2881 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     2941 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     3001 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     3061 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     3121 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     3181 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     3241 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     3301 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     3361 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     3421 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     3481 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     3541 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     3601 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     3661 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     3721 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     3781 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     3841 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     3901 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     3961 gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc
     4021 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa
     4081 ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc
     4141 gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg
     4201 attacgccaa gcttgcatgc atttaaatca ttacgaccga gattcccggg taataactga
     4261 tataattaaa ttgaagctct aatttgtgag tttagtatac atgcatttac ttataataca
     4321 gttttttagt tttgctggcc gcatcttctc aaatatgctt cccagcctgc ttttctgtaa
     4381 cgttcaccct ctaccttagc atcccttccc tttgcaaata gtcctactag taaataatat
     4441 ccttctcgaa agctttaacg aacgcagaat tttcgagtta ttaaacttaa aatacgctga
     4501 acccgaacat agaaatatcg aatgggaaaa aaaaactgca taaaggcatt aaaagaggag
     4561 cgaatttttt tttaataaaa atcttaataa tcattaaaag ataaataata gtctatatat
     4621 acgtatataa ataaaaaata ttcaaaaaat aaaataaact attattttag cgtaaaggat
     4681 ggggaaagag aaaagaaaaa aattgatcta tcgatttcaa ttcaattcaa tttaagatct
     4741 tttgtacaat tcatccatac catgggtaat accagcagca gtaacaaatt ctaacaagac
     4801 catgtggtct ctcttttcgt ttggatcttt ggataaggca gattgagtgg ataagtaatg
     4861 gttgtctggt aacaagactg gaccatcacc aattggagta ttttgttgat aatggtcagc
     4921 taattgaaca gaaccatctt caatgttgtg tctaattttg aagttaactt tgataccatt
     4981 cttttgtttg tcagccatga tgtaaacatt gtgagagtta tagttgtatt ccaatttgtg
     5041 acctaaaatg ttaccatctt ctttaaaatc aatacctttt aattcgattc tattaactaa
     5101 ggtatcacct tcaaacttga cttcagctct ggtcttgtag ttaccgtcat ctttgaaaaa
     5161 aatagttctt tcttgaacat aaccttctgg catggcagac ttgaaaaagt catgttgttt
     5221 catatgatct gggtatctcg caaaacattg aacaccataa ccgaaagtag tgactaaggt
     5281 tggccatgga actggcaatt taccagtagt acaaataaat tttaaggtca atttaccgta
     5341 agtagcatca ccttcacctt caccggagac agaaaatttg tgaccattaa catcaccatc
     5401 taattcaacc aaaattggga caataccagt gaataattct tcacctttag acattttgga
     5461 tcctttgtaa ttaaaactta gattagattg ctatgctttc tttctaatga gcaagaagta
     5521 aaaaaagttg taatagaaca agaaaaatga aactgaaact tgagaaattg aagaccgttt
     5581 attaacttaa atatcaatgg gaggtcatcg aaagagaaaa aaatcaaaaa aaaaaatttt
     5641 caagaaaaag aatctgctcg aggattgccg gcactctatc aatgatagag ttaagccctc
     5701 tctatcactg atagggatac cgcccccgct tatctctatc actgataggg ataccaatta
     5761 ctctatcaat gatagagtta gggcccaatc ttagattcag gtagaacgtg ataaaaattt
     5821 ttattgcctt
//
