LOCUS       sequence-327731-        8553 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gal-inducible yeast expression of the mitochondrial ""clogger"" 
            protein b2(167)-DHFR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8553)
  AUTHORS   Boos F, Kramer L, Groh C, Jung F, Haberkant P, Stein F, Wollweber F,
            Gackstatter A, Zoller E, van der Laan M, Savitski MM, Benes V, 
            Herrmann JM
  TITLE     Mitochondrial protein-induced stress triggers a global adaptive 
            transcriptional programme.
  JOURNAL   Nat Cell Biol. 2019 Apr;21(4):442-451. doi: 
            10.1038/s41556-019-0294-5. Epub 2019 Mar 18.
  PUBMED    30886345
REFERENCE   2  (bases 1 to 8553)
  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/s41556-019-0294-5"; journalName: "Nat Cell Biol"; date: 
            "2019-04"; volume: "21"; issue: "4"; pages: "442-451"
COMMENT     Sequence Label: pYX233-b2-DHFR
FEATURES             Location/Qualifiers
     source          1..8553
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..665
                     /gene="GAL1 and GAL10 genes of S. cerevisiae"
                     /label=GAL1,10 promoter
                     /note="divergent inducible promoter, regulated by Gal4"
     protein_bind    217..334
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     629..652
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     CDS             1204..1767
                     /codon_start=1
                     /gene="Mus musculus Dhfr"
                     /product="mouse dihydrofolate reductase"
                     /label=DHFR
                     /translation="MVRPLNCIVAVSQNMGIGKNGDLPWPPLRNEFKYFQRMTTTSSVE
                     GKQNLVIMGRKTWFSIPEKNRPLKDRINIVLSRELKEPPRGAHFLAKSLDDALRLIEQP
                     ELASKVDMVWIVGGSSVYQEAMNQPGHLRLFVTRIMQEFESDTFFPEIDLGKYKLLPEY
                     PGVLSEVQEEKGIKYKFEVYEKKD"
     CDS             1878..1904
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(2032..2051)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2033..2051)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2058..2075)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2058..2074)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2067..2089)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2215..2670
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2302..2321)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2512..2533
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2773..3447)
                     /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(3448..3729)
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     primer_bind     complement(3750..3769)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3869..3891
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3929..3947)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     4009..4025
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4009..4025
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     rep_origin      4585..5927
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(5548..5595)
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     promoter        6204..6308
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6309..6377,6378..7169)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFVAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(6527..6546)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7340..7928
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7829..7848
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8082..8099
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    8216..8237
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(8252..8257,8258..8275,8276..8282)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8290..8306
                     /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     8295..8317
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     8314..8330
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     8314..8330
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        8351..8369
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 tttcaaaaat tcttactttt tttttggatg gacgcaaaga agtttaataa tcatattaca
       61 tggcattacc accatataca tatccatata catatccata tctaatctta cttatatgtt
      121 gtggaaatgt aaagagcccc attatcttag cctaaaaaaa ccttctcttt ggaactttca
      181 gtaatacgct taactgctca ttgctatatt gaagtacgga ttagaagccg ccgagcgggt
      241 gacagccctc cgaaggaaga ctctcctccg tgcgtcctcg tcttcaccgg tcgcgttcct
      301 gaaacgcaga tgtgcctcgc gccgcactgc tccgaacaat aaagattcta caatactagc
      361 ttttatggtt atgaagagga aaaattggca gtaacctggc cccacaaacc ttcaaatgaa
      421 cgaatcaaat taacaaccat aggatgataa tgcgattagt tttttagcct tatttctggg
      481 gtaattaatc agcgaagcga tgatttttga tctattaaca gatatataaa tgcaaaaact
      541 gcataaccac tttaactaat actttcaaca ttttcggttt gtattacttc ttattcaaat
      601 gtaataaaag tatcaacaaa aaattgttaa tatacctcta tactttaacg tcaaggagaa
      661 aaaaccccgg atcgaattcc caaacaaaag tagtcaatgc taaaatacaa acctttacta
      721 aaaatctcga agaactgtga ggctgctatc ctcagagcgt ctaagactag attgaacaca
      781 atccgcgcgt acggttctac cgttccaaaa tccaagtcgt tcgaacaaga ctcaagaaaa
      841 cgcacacagt catggactgc cttgagagtc ggtgcaattc tagccgctac tagttccgtg
      901 gcgtatctaa actggcataa tggccaaata gacaacgagc cgaaactgga tatgaataaa
      961 caaaagattt cgcccgctga agttgccaag cataacaagc ccgatgattg ttgggttgtg
     1021 atcaatggtt acgtatacga cttaacgcga ttcctaccaa atcatccagg tgggcaggat
     1081 gttatcaagt ttaatgccgg gaaagatgtc actgctattt ttgaaccatt acatgctcct
     1141 aatgtcatcg ataagtatat agctcccgag aaaaaattgg gtccccttca aggatccggc
     1201 atcatggttc gaccattgaa ctgcatcgtc gccgtgtccc aaaatatggg gattggcaag
     1261 aacggagacc taccctggcc tccgctcagg aacgagttca agtacttcca aagaatgacc
     1321 acaacctctt cagtggaagg taaacagaat ctggtgatta tgggtaggaa aacctggttc
     1381 tccattcctg agaagaatcg acctttaaag gacagaatta atatagttct cagtagagaa
     1441 ctcaaagaac caccacgagg agctcatttt cttgccaaaa gtttggatga tgccttaaga
     1501 cttattgaac aaccggaatt ggcaagtaaa gtagacatgg tttggatagt cggaggcagt
     1561 tctgtttacc aggaagccat gaatcaacca ggccacctca gactctttgt gacaaggatc
     1621 atgcaggaat ttgaaagtga cacgtttttc ccagaaattg atttggggaa atataaactt
     1681 ctcccagaat acccaggcgt cctctctgag gtccaggagg aaaaaggcat caagtataag
     1741 tttgaagtct acgagaagaa agactaacag gaagatgctt tcaagttctc tgctcccctc
     1801 ctaaagctat gcatttttat aagaccatgg gacttttgct ggctttagat ccggccaagc
     1861 ttacgcgtcg acccgggtat ccgtatgatg tgcctgacta cgcatgatat ctcgagctca
     1921 gctagctaac tgaataagga acaatgaacg tttttccttt ctcttgttcc tagtattaat
     1981 gactgaccga tacatccctt tttttttttg tctttgtcta gctccaattc gccctatagt
     2041 gagtcgtatt acaattcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc
     2101 gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa
     2161 gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atggacgcgc
     2221 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     2281 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     2341 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     2401 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
     2461 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     2521 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     2581 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     2641 attttaacaa aatattaacg cttacaattt cctgatgcgg tattttctcc ttacgcatct
     2701 gtgcggtatt tcacaccgca tagatcggca agtgcacaaa caatacttaa ataaatacta
     2761 ctcagtaata acctatttct tagcattttt gacgaaattt gctattttgt tagagtcttt
     2821 tacaccattt gtctccacac ctccgcttac atcaacacca ataacgccat ttaatctaag
     2881 cgcatcacca acattttctg gcgtcagtcc accagctaac ataaaatgta aactttcggg
     2941 gctctcttgc cttccaaccc agtcagaaat cgagttccaa tccaaaagtt cacctgtccc
     3001 acctgcttct gaatcaaaca agggaataaa cgaatgaggt ttctgtgaag ctgcactgag
     3061 tagtatgttg cagtcttttg gaaatacgag tcttttaata actggcaaac cgaggaactc
     3121 ttggtattct tgccacgact catctccatg cagttggact atatcaatgc cgtaatcatt
     3181 gaccagagcc aaaacatcct ccttaggttg attacgaaac acgccaacca agtatttcgg
     3241 agtgcctgaa ctatttttat atgcttttac aagacttgaa attttccttg caataaccgg
     3301 gtcaattgtt ctctttctat tgggcacaca tataataccc agcaagtcag catcggaatc
     3361 tagagcacat tctgcggcct ctgtgctctg caagccgcaa actttcacca atggaccaga
     3421 actacctgtg aaattaataa cagacatact ccaagctgcc tttgtgtgct taatcacgta
     3481 tactcacgtg ctcaatagtc accaatgccc tccctcttgg ccctctcctt ttcttttttc
     3541 gaccgaatta attcttaatc ggcaaaaaaa gaaaagctcc ggatcaagat tgtacgtaag
     3601 gtgacaagct atttttcaat aaagaatatc ttccactact gccatctggc gtcataactg
     3661 caaagtacac atatattacg atgctgttct attaaatgct tcctatatta tatatatagt
     3721 aatgtcgttg atctatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     3781 cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     3841 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     3901 tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat
     3961 gtcatgataa taatggtttc ttagacgtac tagtgagagt gcggccgctc tagaactagt
     4021 ggatcaattc cacggactat agactatact agtatactcc gtctactgta cgatacactt
     4081 ccgctcaggt ccttgtcctt taacgaggcc ttaccactct tttgttactc tattgatcca
     4141 gctcagcaaa ggcagtgtga tctaagattc tatcttcgcg atgtagtaaa actagctaga
     4201 ccgagaaaga gactagaaat gcaaaaggca cttctacaat ggctgccatc attattatcc
     4261 gatgtgacgc tgcagcttct caatgatatt cgaatacgct ttgaggagat acagcctaat
     4321 atccgacaaa ctgttttaca gatttacgat cgtacttgtt acccatcatt gaattttgaa
     4381 catccgaacc tgggagtttt ccctgaaaca gatagtatat ttgaacctgt ataataatat
     4441 atagtctagc gctttacgga agacaatgta tgtatttcgg ttcctggaga aactattgca
     4501 tctattgcat aggtaatctt gcacgtcgca tccccggttc attttctgcg tttccatctt
     4561 gcacttcaat agcatatctt tgttaacgaa gcatctgtgc ttcattttgt agaacaaaaa
     4621 tgcaacgcga gagcgctaat ttttcaaaca aagaatctga gctgcatttt tacagaacag
     4681 aaatgcaacg cgaaagcgct attttaccaa cgaagaatct gtgcttcatt tttgtaaaac
     4741 aaaaatgcaa cgcgagagcg ctaatttttc aaacaaagaa tctgagctgc atttttacag
     4801 aacagaaatg caacgcgaga gcgctatttt accaacaaag aatctatact tcttttttgt
     4861 tctacaaaaa tgcatcccga gagcgctatt tttctaacaa agcatcttag attacttttt
     4921 ttctcctttg tgcgctctat aatgcagtct cttgataact ttttgcactg taggtccgtt
     4981 aaggttagaa gaaggctact ttggtgtcta ttttctcttc cataaaaaaa gcctgactcc
     5041 acttcccgcg tttactgatt actagcgaag ctgcgggtgc attttttcaa gataaaggca
     5101 tccccgatta tattctatac cgatgtggat tgcgcatact ttgtgaacag aaagtgatag
     5161 cgttgatgat tcttcattgg tcagaaaatt atgaacggtt tcttctattt tgtctctata
     5221 tactacgtat aggaaatgtt tacattttcg tattgttttc gattcactct atgaatagtt
     5281 cttactacaa tttttttgtc taaagagtaa tactagagat aaacataaaa aatgtagagg
     5341 tcgagtttag atgcaagttc aaggagcgaa aggtggatgg gtaggttata tagggatata
     5401 gcacagagat atatagcaaa gagatacttt tgagcaatgt ttgtggaagc ggtattcgca
     5461 atattttagt agctcgttac agtccggtgc gtttttggtt ttttgaaagt gcgtcttcag
     5521 agcgcttttg gttttcaaaa gcgctctgaa gttcctatac tttctagaga ataggaactt
     5581 cggaatagga acttcaaagc gtttccgaaa acgagcgctt ccgaaaatgc aacgcgagct
     5641 gcgcacatac agctcactgt tcacgtcgca cctatatctg cgtgttgcct gtatatatat
     5701 atacatgaga agaacggcat agtgcgtgtt tatgcttaaa tgcgtactta tatgcgtcta
     5761 tttatgtagg atgaaaggta gtctagtacc tcctgtgata ttatcccatt ccatgcgggg
     5821 tatcgtatgc ttccttcagc actacccttt agctgttcta tatgctgcca ctcctcaatt
     5881 ggattagtct catccttcaa tgctatcatt tcctttgata ttggatcata tgcatagtac
     5941 cgagaaacta gtgcgaagta gtgatcaggt attgctgtta tctgatgagt atacgttgtc
     6001 ctggccacgg cagaagcacg cttatcgctc caatttccca caacattagt caactccgtt
     6061 aggcccttca ttgaaagaaa tgaggtcatc aaatgtcttc caatgtgaga ttttgggcca
     6121 ttttttatag caaagattga ataaggcgca tttttcttca aagctgcggc cgcacgtcag
     6181 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     6241 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     6301 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gtggcatttt
     6361 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     6421 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     6481 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     6541 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     6601 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     6661 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     6721 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     6781 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     6841 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     6901 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     6961 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     7021 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     7081 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     7141 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     7201 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     7261 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     7321 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     7381 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     7441 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     7501 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     7561 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     7621 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     7681 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     7741 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     7801 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     7861 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     7921 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     7981 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     8041 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     8101 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
     8161 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg
     8221 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt
     8281 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg
     8341 ccaagctcga aattaaccct cactaaaggg aacaaaagct ggtaccgggc cggccgtcgg
     8401 accaattcga caggttatca gcaacaacac agtcatatcc attctcaatt agctctacca
     8461 cagtgtgtga accaatgtat ccagcaccac ctgtaaccaa aacaatttta gaagtacttt
     8521 cactttgtaa ctgagctgtc atttatattg aat
//
