LOCUS       sequence-370754-        8987 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expressing artificial trans-repressor in Saccharomyces cerevisiae.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8987)
  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 8987)
  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: pAF9D94 (pAF10C6)
FEATURES             Location/Qualifiers
     source          1..8987
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(193..212)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(194..212)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(222..239)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(222..238)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(231..253)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      379..834
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(466..485)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     676..697
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(937..1611)
                     /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(1612..1892)
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     primer_bind     complement(1913..1932)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2032..2054
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2092..2110)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    2151..2654
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        2691..2795
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2796..2864,2865..3656)
                     /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(3014..3033)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3827..4415
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4316..4335
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4569..4586
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4703..4724
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4739..4744,4745..4762,4763..4769)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4777..4793
                     /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     4782..4804
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4801..4817
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4801..4817
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     4836..4856
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        4838..4856
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     CDS             6061..6081
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
ORIGIN
        1 tgcaggatac ccagttgaga agacgagcta tttttctcat gtttcttttg tttattcact
       61 tttctatact ttttcttttc tattgaaata tatattatat cttatgtatg tttttttgta
      121 ttataaacaa gcaaatttcg tcttgccatt taccttctga tattaatcac atgtatatcg
      181 ggccccaatt cgccctatag tgagtcgtat tacgcgcgct cactggccgt cgttttacaa
      241 cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct
      301 ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc
      361 agcctgaatg gcgaatggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
      421 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
      481 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
      541 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
      601 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
      661 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
      721 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
      781 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttcctgat
      841 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatagatc ggcaagtgca
      901 caaacaatac ttaaataaat actactcagt aataacctat ttcttagcat ttttgacgaa
      961 atttgctatt ttgttagagt cttttacacc atttgtctcc acacctccgc ttacatcaac
     1021 accaataacg ccatttaatc taagcgcatc accaacattt tctggcgtca gtccaccagc
     1081 taacataaaa tgtaagcttt cggggctctc ttgccttcca acccagtcag aaatcgagtt
     1141 ccaatccaaa agttcacctg tcccacctgc ttctgaatca aacaagggaa taaacgaatg
     1201 aggtttctgt gaagctgcac tgagtagtat gttgcagtct tttggaaata cgagtctttt
     1261 aataactggc aaaccgagga actcttggta ttcttgccac gactcatctc catgcagttg
     1321 gacgatatca atgccgtaat cattgaccag agccaaaaca tcctccttag gttgattacg
     1381 aaacacgcca accaagtatt tcggagtgcc tgaactattt ttatatgctt ttacaagact
     1441 tgaaattttc cttgcaataa ccgggtcaat tgttctcttt ctattgggca cacatataat
     1501 acccagcaag tcagcatcgg aatccaaagc acattctgcg gcctctgtgc tctgcaagcc
     1561 gcaaactttc accaatggac cagaactacc tgtgaaatta ataacagaca tactccaagc
     1621 tgcctttgtg tgcttaatca cgtatactca cgtgctcaat agtcaccaat gccctccctc
     1681 ttggccctct ccttttcttt tttcgaccga attaattctt aatcggcaaa aaaagaaaag
     1741 ctccggatca agattgtacg taaggtgaca agctattttt caataaagaa tatcttccac
     1801 tactgccatc tggcgtcata actgcaaagt acacatatat tacgatgctg ttctattaaa
     1861 tgcttcctat attatatata tagtaatgtc gtgatctatg gtgcactctc agtacaatct
     1921 gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct
     1981 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
     2041 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga
     2101 tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg gatcgcttgc
     2161 ctgtaactta cacgcgcctc gtatctttta atgatggaat aatttgggaa tttactctgt
     2221 gtttatttat ttttatgttt tgtatttgga ttttagaaag taaataaaga aggtagaaga
     2281 gttacggaat gaagaaaaaa aaataaacaa aggtttaaaa aatttcaaca aaaagcgtac
     2341 tttacatata tatttattag acaagaaaag cagattaaat agatatacat tcgattaacg
     2401 ataagtaaaa tgtaaaatca caggattttc gtgtgtggtc ttctacacag acaagatgaa
     2461 acaattcggc attaatacct gagagcagga agagcaagat aaaaggtagt atttgttggc
     2521 gatcccccta gagtctttta catcttcgga aaacaaaaac tattttttct ttaatttctt
     2581 tttttacttt ctatttttaa tttatatatt tatattaaaa aatttaaatt ataattattt
     2641 ttatagcacg tgatgaaaag gacccaggtg gcacttttcg gggaaatgtg cgcggaaccc
     2701 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     2761 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     2821 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     2881 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     2941 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     3001 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac
     3061 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     3121 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     3181 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     3241 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     3301 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc
     3361 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     3421 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     3481 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc
     3541 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     3601 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     3661 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     3721 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     3781 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     3841 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     3901 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     3961 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     4021 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     4081 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     4141 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     4201 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     4261 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     4321 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     4381 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     4441 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     4501 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
     4561 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc
     4621 tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag
     4681 cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt
     4741 tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca
     4801 caggaaacag ctatgaccat gattacgcca agcgcgcaat taaccctcac taaagggaac
     4861 aaaagctgga gctctacaat gaaaacaaca ccaagctgcc aaaagctgta tattaaagtt
     4921 aaagctccct catttttagt aaggacgctt tcttttccca caaaagggac gaggaaaaaa
     4981 agaaaaacct ggaaatatag tcacgtgaca tgaataaaca agacctaggg caatatggaa
     5041 aaagaagcta ggataaaaat acatttatga gggttgtaag gcaaagtggc tcagcattaa
     5101 gtagggctaa aaagagtcgc aaatatacat cgtaccgagt gatgaacgtg gttttgaaca
     5161 ccttgttctc ttttgttctc gctccctaca ttcactatat attagaagaa atttcacctc
     5221 aatggacaac tcgagaaatg aatacagaaa tatgtttttt agcgaaattt tcctttcttc
     5281 ttgtcttctt gttttattta aacttccaag gctttaactc agtgtcaaac ataacaacct
     5341 cctcctcccc cacctacgac aacaaccgcc actccgcgga tcatgtccag attggataag
     5401 tccaaggtta ttaactctgc cttggaattg ttgaacgaag ttggtattga aggtttgacc
     5461 actagaaagt tggctcaaaa attgggtgtt gaacaaccta ccttgtactg gcatgttaag
     5521 aacaaaagag ctttgttgga cgctttggcc attgaaatgt tggatagaca tcatacccat
     5581 ttctgtccat tggaaggtga atcatggcaa gatttcttga gaaacaacgc caagtctttc
     5641 agatgtgctt tgttgtctca tagagatggt gctaaagttc acttgggtac tagaccaact
     5701 gaaaagcaat acgaaacttt ggaaaaccag ttggctttct tgtgtcaaca aggtttctct
     5761 ttagaaaacg ccttgtatgc tttgtctgct gttggtcatt ttaccttggg ttgtgttttg
     5821 gaagatcaag aacatcaagt cgccaaagaa gaaagggaaa ctccaactac tgattctatg
     5881 ccaccattat tgagacaagc cattgagttg tttgatcatc aaggtgctga accagctttt
     5941 ttgtttggtt tggaattgat catctgcggt ttggagaaac agttgaaatg tgaatctggc
     6001 tccggtaccg gtggtggtac tccagctgct gcttctactt tggaatctag atgtacccca
     6061 cctaagaaga aaagaaaggt tgctagcaat ccgggcggtg aacaaacaat aatggaacaa
     6121 cccgctcaac agcaacaaca acagcaacaa caacagcagc aacagcaaca gcaggcagca
     6181 gttcctcagc agccactcga cccattaaca caatcaactg cggaaacttg gctctccatt
     6241 gcttctttgg cagaaaccct tggtgatggc gacagggccg caatggcata tgacgccact
     6301 ttacagttca atccctcatc tgcaaaggct ttaacatctt tggctcactt gtaccgttcc
     6361 agagacatgt tccaaagagc tgcagaatta tatgaaagag cacttttggt aaatcccgaa
     6421 ctatcagatg tgtgggctac tttaggtcat tgttatctga tgctggatga tctgcaaaga
     6481 gcttacaatg cctatcaaca ggctctctac cacctcagta atcccaacgt accgaaatta
     6541 tggcatggaa tcggcattct ttatgacaga tatggttcgc tcgactatgc cgaagaagct
     6601 tttgccaaag ttttggaatt ggaccctcat tttgaaaagg caaacgaaat ttacttcaga
     6661 ctaggtatta tttataaaca tcagggtaaa tggtctcaag ctttggaatg cttcagatac
     6721 attatccctc aacctcctgc tcccttgcag gagtgggaca tatggtttca gttgggtagt
     6781 gttttggaga gtatgggaga gtggcaaggt gcgaaggaag cctacgagca tgtcttggct
     6841 caaaatcaac atcatgccaa agtattacaa caattaggtt gtctttacgg tatgagtaac
     6901 gtacaatttt atgaccctca aaaggcattg gattatcttc taaagtcgtt agaagcagat
     6961 ccctccgatg ccactacatg gtaccatctc ggtagagtgc atatgattag aacagattat
     7021 actgccgcat atgatgcttt ccaacaagct gttaatagag attcaagaaa ccctatcttt
     7081 tggtgctcaa tcggtgtttt atattaccaa atttctcaat acagagacgc cttagacgcg
     7141 tacacaagag ccataagatt aaatccttat attagtgaag tttggtacga tctaggtact
     7201 ctttacgaaa cttgtaacaa ccaattatct gacgcccttg atgcgtataa gcaagctgca
     7261 agactggacg taaataatgt tcacataaga gaaagattag aagctttaac aaagcagtta
     7321 gaaaacccag gcaatataaa caaatcgaac ggtgcgccaa cgaatgcctc tcctgcccca
     7381 cctcctgtga ttttacaacc taccttacaa cctaatgatc aaggaaatcc tttgaacact
     7441 agaatttcag cccaatctgc caatgctact gcttcaatgg tacaacaaca gcatcctgct
     7501 caacaaacgc ctattaactc ttctgcaaca atgtacagta atggagcttc ccctcaatta
     7561 caagctcaag ctcaagctca agctcaagca caagctcaag cacaagcaca agctcaagca
     7621 caagcacaag cacaagcgca agcacaagca caagcacagg cgcaagcaca ggcacaagca
     7681 caagcacaag cacatgcaca agcgcaagca caagcacaag cacaggcaca agcacaagca
     7741 caggcgcagg cacaacaaca acaacaacaa cagcaacaac aacaacaaca acaacaacaa
     7801 caacaacaac aacaacaaca acaacaacaa caacagcagc agcaattaca gcccctacca
     7861 agacaacagc tgcagcaaaa gggagtttct gtgcaaatgt taaatcctca acaagggcaa
     7921 ccatatatca cacagccaac agtcatacaa gctcaccaac tgcaaccatt ttctacacaa
     7981 gctatggaac atccgcaaag ctctcaactg ccacctcaac agcaacaact acaatctgtt
     8041 caacatccac aacaacttca aggccagcct caagcccaag ctccccaacc tttaatccag
     8101 cataacgtgg aacagaacgt tttacctcaa aagagataca tggaaggtgc aatccacact
     8161 ttagtagatg ccgccgtatc cagtagcacc cacacagaga ataacacaaa gtctcctcgt
     8221 caaccaaccc atgccattcc aacgcaagct cccgcaacag gaataacgaa cgctgaacca
     8281 caggtaaaga agcaaaagtt gaactctcca aattcaaaca tcaacaaatt agtaaatact
     8341 gctacttcca ttgaagaaaa tgcaaaatct gaggtgagca accaatcgcc agcagtagtg
     8401 gagtctaata ccaataatac ttcacaagaa gaaaaacctg taaaagcaaa ctcaatacct
     8461 tcagtaattg gcgcacagga acctccacag gaagctagtc ctgctgaaga agctaccaaa
     8521 gcagcttctg tttctccttc tacaaaaccg cttaatacgg aaccagagtc atctagtgtc
     8581 caaccaactg tatcatcaga aagttcaaca acaaaagcaa atgaccaaag cactgctgag
     8641 accatagaac tttctactgc tactgttcct gcagaagcaa gccctgtaga agacgaagta
     8701 agacagcatt ctaaagagga aaacggcaca actgaagcat ctgcaccttc tactgaagag
     8761 gcggagccag cagcttccag agatgctgaa aaacaacaag atgaaaccgc tgctacaacg
     8821 ataactgtaa tcaaacctac tttggaaaca atggaaacag tgaaagagga ggccaaaatg
     8881 cgtgaggaag agcaaacatc tcaagaaaaa tccccacagg agaacacact tccaagagaa
     8941 aatgtagtaa ggcaagtgga agaagatgaa aactacgacg actaacc
//
