LOCUS       sequence-370758-        6917 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 6917)
  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 6917)
  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: pAFZifPZ431
FEATURES             Location/Qualifiers
     source          1..6917
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             353..373
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             954..965
                     /codon_start=1
                     /product="WELQut protease recognition and cleavage site"
                     /label=WELQut site
                     /translation="WELQ"
     primer_bind     complement(1765..1784)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1766..1784)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1794..1811)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1794..1810)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1803..1825)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      1951..2406
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2038..2057)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2248..2269
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2509..3183)
                     /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"
     promoter        complement(3184..3464)
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     primer_bind     complement(3485..3504)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3604..3626
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3664..3682)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    3723..4226
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        4263..4367
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4368..4436,4437..5228)
                     /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(4586..4605)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5399..5987
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5888..5907
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6141..6158
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6275..6296
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6311..6316,6317..6334,6335..6341)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6349..6365
                     /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     6354..6376
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6373..6389
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6373..6389
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     6408..6428
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        6410..6428
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 tcccccacct acgacaacaa ccgccactcc gcggatcatg ggtactagac catacgcttt
       61 taagtgtcca gtttgtggta aagccttcag acattcttca tccttggtta gacatcaaag
      121 aactcatact ggtcaaaagc catacaagtg taagcaatgt ggaaaggctt ttggttgtcc
      181 atctaatttg agaaggcatg gtagaaccca tacaggtgaa aaaccttatg aatgtcacga
      241 ttgtggcaag tctttcagac aatctactca tttgactcag catagaagaa tccacaccgg
      301 tggtggtggt actccagctg ctgcttctac tttggaatct agatgtaccc cacctaagaa
      361 gaaaagaaag gttgctagcg actcgattat accggcaggc gtcaagctag atgatttaca
      421 agtgatattg gctaagaacg agaatgaaac cagggacaaa gtgtgtaagc agatcaacga
      481 agcacgcgat gaaattctgc cgttgcgatt gcagttcaat gaattcatac agataatggc
      541 aaacatagac caagaaggat ccaagcaggc tgaccgtatg gccaaatacc tacatatcag
      601 agacaagatt ttgcaactga acgatagatt ccagactttg tcttcgcatt tagaagcact
      661 gcaaccttta ttcagcactg taccagaata cttgaagact gcagataaca gggacagaag
      721 tttccagctt ttggaaccgt tgagtactta taacaaaaat ggcaacgccg tttgctcgac
      781 agcaactgtt gtaagcacca accactctgc ggccgcttcg acacccacca caactgccac
      841 ccctcatgca aatcccatca cacatgcgca ctcgctttcg aatccaaata gcaccgctac
      901 aatgcaacat aatcccctgg ccggcaagag aggtccaaaa agcggtagta ctatgggaac
      961 tccaaccgta cataatagca ctgcagctgc accgattgcg gcacctaaaa agccaagaaa
     1021 accaagacaa accaagaagg caaaggctca agctcaggct caagcgcagg ctcaagcgca
     1081 ggtctacgca cagcagtcga ctgtccagac gccaataaca gcatcgatgg cagccgcgct
     1141 acccaaccca actcctagca tgattaacag cgtttcgccc acaaatgtta tgggcacgcc
     1201 gctgaccaac atgatgtctc ccatggggaa cgcatactca atgggagctc aaaaccaagg
     1261 gggacaagta tctatgtcac aattcaatgg cagcggcaat ggatccaatc caaatacaaa
     1321 tacaaactcc aataacaccc cgctacagtc acaattaaac ctaaacaatc taacccctgc
     1381 taatatccta aatatgagca tgaataacga tttccagcag cagcagcaac agcaacagca
     1441 gcagcagcag cctcaaccac agtacaatat gaatatgggc atgaataaca tgaataacgg
     1501 ggggaaagaa ctggattctc tagacctgaa caatctggaa ttaggtggtc tgaacatgga
     1561 tttcttgtaa cctgcaggat acccagttga gaagacgagc tatttttctc atgtttcttt
     1621 tgtttattca cttttctata ctttttcttt tctattgaaa tatatattat atcttatgta
     1681 tgtttttttg tattataaac aagcaaattt cgtcttgcca tttaccttct gatattaatc
     1741 acatgtatat cgggccccaa ttcgccctat agtgagtcgt attacgcgcg ctcactggcc
     1801 gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca
     1861 gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc
     1921 caacagttgc gcagcctgaa tggcgaatgg acgcgccctg tagcggcgca ttaagcgcgg
     1981 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
     2041 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     2101 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     2161 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     2221 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     2281 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
     2341 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta
     2401 caatttcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcataga
     2461 tcggcaagtg cacaaacaat acttaaataa atactactca gtaataacct atttcttagc
     2521 atttttgacg aaatttgcta ttttgttaga gtcttttaca ccatttgtct ccacacctcc
     2581 gcttacatca acaccaataa cgccatttaa tctaagcgca tcaccaacat tttctggcgt
     2641 cagtccacca gctaacataa aatgtaagct ttcggggctc tcttgccttc caacccagtc
     2701 agaaatcgag ttccaatcca aaagttcacc tgtcccacct gcttctgaat caaacaaggg
     2761 aataaacgaa tgaggtttct gtgaagctgc actgagtagt atgttgcagt cttttggaaa
     2821 tacgagtctt ttaataactg gcaaaccgag gaactcttgg tattcttgcc acgactcatc
     2881 tccatgcagt tggacgatat caatgccgta atcattgacc agagccaaaa catcctcctt
     2941 aggttgatta cgaaacacgc caaccaagta tttcggagtg cctgaactat ttttatatgc
     3001 ttttacaaga cttgaaattt tccttgcaat aaccgggtca attgttctct ttctattggg
     3061 cacacatata atacccagca agtcagcatc ggaatccaaa gcacattctg cggcctctgt
     3121 gctctgcaag ccgcaaactt tcaccaatgg accagaacta cctgtgaaat taataacaga
     3181 catactccaa gctgcctttg tgtgcttaat cacgtatact cacgtgctca atagtcacca
     3241 atgccctccc tcttggccct ctccttttct tttttcgacc gaattaattc ttaatcggca
     3301 aaaaaagaaa agctccggat caagattgta cgtaaggtga caagctattt ttcaataaag
     3361 aatatcttcc actactgcca tctggcgtca taactgcaaa gtacacatat attacgatgc
     3421 tgttctatta aatgcttcct atattatata tatagtaatg tcgtgatcta tggtgcactc
     3481 tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg
     3541 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     3601 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa
     3661 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga
     3721 cggatcgctt gcctgtaact tacacgcgcc tcgtatcttt taatgatgga ataatttggg
     3781 aatttactct gtgtttattt atttttatgt tttgtatttg gattttagaa agtaaataaa
     3841 gaaggtagaa gagttacgga atgaagaaaa aaaaataaac aaaggtttaa aaaatttcaa
     3901 caaaaagcgt actttacata tatatttatt agacaagaaa agcagattaa atagatatac
     3961 attcgattaa cgataagtaa aatgtaaaat cacaggattt tcgtgtgtgg tcttctacac
     4021 agacaagatg aaacaattcg gcattaatac ctgagagcag gaagagcaag ataaaaggta
     4081 gtatttgttg gcgatccccc tagagtcttt tacatcttcg gaaaacaaaa actatttttt
     4141 ctttaatttc tttttttact ttctattttt aatttatata tttatattaa aaaatttaaa
     4201 ttataattat ttttatagca cgtgatgaaa aggacccagg tggcactttt cggggaaatg
     4261 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     4321 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     4381 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     4441 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     4501 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     4561 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg
     4621 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     4681 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     4741 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     4801 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     4861 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg
     4921 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     4981 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     5041 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg
     5101 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     5161 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     5221 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     5281 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     5341 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     5401 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     5461 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     5521 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
     5581 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     5641 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     5701 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     5761 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     5821 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     5881 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     5941 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     6001 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     6061 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
     6121 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac
     6181 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc
     6241 ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg
     6301 caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat
     6361 aacaatttca cacaggaaac agctatgacc atgattacgc caagcgcgca attaaccctc
     6421 actaaaggga acaaaagctg gagctctaca atgaaaacaa caccaagctg ccaaaagctg
     6481 tatattaaag ttaaagctcc ctcattttta gtaaggacgc tttcttttcc cacaaaaggg
     6541 acgaggaaaa aaagaaaaac ctggaaatat agtcacgtga catgaataaa caagacctag
     6601 ggcaatatgg aaaaagaagc taggataaaa atacatttat gagggttgta aggcaaagtg
     6661 gctcagcatt aagtagggct aaaaagagtc gcaaatatac atcgtaccga gtgatgaacg
     6721 tggttttgaa caccttgttc tcttttgttc tcgctcccta cattcactat atattagaag
     6781 aaatttcacc tcaatggaca actcgagaaa tgaatacaga aatatgtttt ttagcgaaat
     6841 tttcctttct tcttgtcttc ttgttttatt taaacttcca aggctttaac tcagtgtcaa
     6901 acataacaac ctcctcc
//
