LOCUS       sequence-368099-        5933 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expressing artificial trans-activator in Saccharomyces cerevisiae.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5933)
  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 5933)
  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: pAF4EH4R
FEATURES             Location/Qualifiers
     source          1..5933
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     231..253
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     245..262
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     246..262
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     272..291
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        272..290
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             complement(699..719)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(1561..1581)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1561..1579)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1600..1616)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1600..1616)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1613..1635)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1624..1640
                     /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(1648..1654,1655..1672,1673..1678))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1693..1714
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1831..1848)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2002..2590)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2082..2101)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2761..3552,3553..3621))
                     /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     3384..3403
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3622..3726)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    3763..4266
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     primer_bind     4307..4325
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4363..4385)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4485..4504
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        4525..4805
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     CDS             4806..5480
                     /codon_start=1
                     /gene="S. cerevisiae TRP1"
                     /product="phosphoribosylanthranilate isomerase, required 
                     for tryptophan biosynthesis"
                     /label=TRP1
                     /note="yeast auxotrophic marker"
                     /translation="MSVINFTGSSGPLVKVCGLQSTEAAECALDSDADLLGIICVPNRK
                     RTIDPVIARKISSLVKAYKNSSGTPKYLVGVFRNQPKEDVLALVNDYGIDIVQLHGDES
                     WQEYQEFLGLPVIKRLVFPKDCNILLSAASQKPHSFIPLFDSEAGGTGELLDWNSISDW
                     VGRQESPESLHFMLAGGLTPENVGDALRLNGVIGVDVSGGVETNGVKDSNKIANFVKNA
                     KK"
     rep_origin      complement(join(5583..5933,1..105))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5720..5741)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     join(5932..5933,1..18)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg
       61 taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtccatt cgccattcag
      121 gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagctggc
      181 gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt cccagtcacg
      241 acgttgtaaa acgacggcca gtgagcgcgc gtaatacgac tcactatagg gcgaattggg
      301 gcccgatata catgtgatta atatcagaag gtaaatggca agacgaaatt tgcttgttta
      361 taatacaaaa aaacatacat aagatataat atatatttca atagaaaaga aaaagtatag
      421 aaaagtgaat aaacaaaaga aacatgagaa aaatagctcg tcttctcaac tgggtatcct
      481 gcaggttaac caccgtattc atcaataccc aaggcatcag taaacatttg ttcaaattca
      541 aaatcagcca tatccaaagc accataagga gcagaatcat gaggagtaaa acctggacct
      601 ggagagtcac cgtcacctaa catatccaag tcaaaatcat ccaaagcatc agcatgagcc
      661 atagcaacat cttcaccatc caaatgcaat tcggtaccaa cctttctttt cttcttaggt
      721 ggggtacagt cgacttccaa agtagaagca gcagctggag taccaccacc tctagaatga
      781 attttggtat gtctttttct ttcgtcggat ctagcaaact ttctaccgca aatatcacaa
      841 gcgaatggtt tttcaccagt gtgggttctg atatgagtag tcaaatgatc agacctggag
      901 aagttcctca tacagattct acattggaat ggcttttgac cggtatggat tctaatatgt
      961 ctggtcaatt catcggatct ggagaatctt ctatcacaag attcaactgg acaagcgtat
     1021 ggtctagtac ccatgatgga tccagtggcg gttgttgtcg taggtggggg aggaggaggt
     1081 tgttatgttt gacactgagt taaagccttg gaagtttaaa taaaacaaga agacaagaag
     1141 aaaggaaaat ttcgctaaaa aacatatttc tgtattcatt tctcgagttg tccattgagg
     1201 tgaaatttct tctaatatat agtgaatgta gggagcgaga acaaaagaga acaaggtgtt
     1261 caaaaccacg ttcatcactc ggtacgatgt atatttgcga ctctttttag ccctacttaa
     1321 tgctgagcca ctttgcctta caaccctcat aaatgtattt ttatcctagc ttctttttcc
     1381 atattgccct aggtcttgtt tattcatgtc acgtgactat atttccaggt ttttcttttt
     1441 ttcctcgtcc cttttgtggg aaaagaaagc gtccttacta aaaatgaggg agctttaact
     1501 ttaatataca gcttttggca gcttggtgtt gttttcattg tagagctcca gcttttgttc
     1561 cctttagtga gggttaattg cgcgcttggc gtaatcatgg tcatagctgt ttcctgtgtg
     1621 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
     1681 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
     1741 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     1801 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     1861 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     1921 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     1981 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     2041 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     2101 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     2161 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     2221 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     2281 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     2341 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     2401 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     2461 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     2521 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     2581 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     2641 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     2701 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     2761 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     2821 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     2881 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     2941 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     3001 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     3061 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     3121 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     3181 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     3241 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     3301 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     3361 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     3421 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     3481 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     3541 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     3601 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     3661 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     3721 tccgcgcaca tttccccgaa aagtgccacc tgggtccttt tcatcacgtg ctataaaaat
     3781 aattataatt taaatttttt aatataaata tataaattaa aaatagaaag taaaaaaaga
     3841 aattaaagaa aaaatagttt ttgttttccg aagatgtaaa agactctagg gggatcgcca
     3901 acaaatacta ccttttatct tgctcttcct gctctcaggt attaatgccg aattgtttca
     3961 tcttgtctgt gtagaagacc acacacgaaa atcctgtgat tttacatttt acttatcgtt
     4021 aatcgaatgt atatctattt aatctgcttt tcttgtctaa taaatatata tgtaaagtac
     4081 gctttttgtt gaaatttttt aaacctttgt ttattttttt ttcttcattc cgtaactctt
     4141 ctaccttctt tatttacttt ctaaaatcca aatacaaaac ataaaaataa ataaacacag
     4201 agtaaattcc caaattattc catcattaaa agatacgagg cgcgtgtaag ttacaggcaa
     4261 gcgatccgtc taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac
     4321 gaggcccttt cgtctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct
     4381 cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg
     4441 cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac tatgcggcat cagagcagat
     4501 tgtactgaga gtgcaccata gatcacgaca ttactatata tataatatag gaagcattta
     4561 atagaacagc atcgtaatat atgtgtactt tgcagttatg acgccagatg gcagtagtgg
     4621 aagatattct ttattgaaaa atagcttgtc accttacgta caatcttgat ccggagcttt
     4681 tctttttttg ccgattaaga attaattcgg tcgaaaaaag aaaaggagag ggccaagagg
     4741 gagggcattg gtgactattg agcacgtgag tatacgtgat taagcacaca aaggcagctt
     4801 ggagtatgtc tgttattaat ttcacaggta gttctggtcc attggtgaaa gtttgcggct
     4861 tgcagagcac agaggccgca gaatgtgctt tggattccga tgctgacttg ctgggtatta
     4921 tatgtgtgcc caatagaaag agaacaattg acccggttat tgcaaggaaa atttcaagtc
     4981 ttgtaaaagc atataaaaat agttcaggca ctccgaaata cttggttggc gtgtttcgta
     5041 atcaacctaa ggaggatgtt ttggctctgg tcaatgatta cggcattgat atcgtccaac
     5101 tgcatggaga tgagtcgtgg caagaatacc aagagttcct cggtttgcca gttattaaaa
     5161 gactcgtatt tccaaaagac tgcaacatac tactcagtgc agcttcacag aaacctcatt
     5221 cgtttattcc cttgtttgat tcagaagcag gtgggacagg tgaacttttg gattggaact
     5281 cgatttctga ctgggttgga aggcaagaga gccccgaaag cttacatttt atgttagctg
     5341 gtggactgac gccagaaaat gttggtgatg cgcttagatt aaatggcgtt attggtgttg
     5401 atgtaagcgg aggtgtggag acaaatggtg taaaagactc taacaaaata gcaaatttcg
     5461 tcaaaaatgc taagaaatag gttattactg agtagtattt atttaagtat tgtttgtgca
     5521 cttgccgatc tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca
     5581 ggaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
     5641 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
     5701 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
     5761 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
     5821 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
     5881 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aag
//
