LOCUS       sequence-176697-        7397 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses GFP1-10 in the mitochondrial matrix.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7397)
  AUTHORS   Ruan L, Zhou C, Jin E, Kucharavy A, Zhang Y, Wen Z, Florens L, Li R
  TITLE     Cytosolic proteostasis through importing of misfolded proteins into 
            mitochondria.
  JOURNAL   Nature. 2017 Mar 16;543(7645):443-446. doi: 10.1038/nature21695. 
            Epub 2017 Mar 1.
  PUBMED    28241148
REFERENCE   2  (bases 1 to 7397)
  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/nature21695"; journalName: "Nature"; date: "2017-03-16- 
            16"; volume: "543"; issue: "7645"; pages: "443-446"
COMMENT     Sequence Label: p404-Grx5-GFP1-10
FEATURES             Location/Qualifiers
     source          1..7397
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(10..28)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        96..200
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(201..269,270..1061)
                     /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(419..438)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1232..1820
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1721..1740
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1974..1991
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        join(2144..2149,2150..2167,2168..2174)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2182..2198
                     /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     2187..2209
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2206..2222
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2206..2222
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     2241..2261
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2243..2261
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     gene            complement(2280..3409)
                     /label=natMX6
                     /note="yeast selectable marker conferring nourseothricin 
                     resistance"
     terminator      complement(2280..2477)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(2493..3065)
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGFT
                     LREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVISYSAWNRR
                     LTVEDIEVAPEHRGHGVGRALMGLATEFAGERGAGHLWLEVTNVNAPAIHAYRRMGFTL
                     CGLDTALYDGTASDGERQALYMSMPCP"
     promoter        complement(3066..3409)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     promoter        3422..4065
                     /gene="S. cerevisiae TDH3"
                     /label=GAP promoter
                     /note="promoter for glyceraldehyde-3-phosphate 
                     dehydrogenase; also known as the TDH3 promoter"
     primer_bind     3959..3982
                     /label=GPDpro-F
                     /note="S. cerevisiae GPD promoter, forward primer"
     CDS             4539..4565
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     terminator      5247..5494
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(5263..5281)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(5512..5531)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5513..5531)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5541..5558)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5541..5557)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5550..5572)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5702..6157
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5789..5808)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5999..6020
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(6256..6930)
                     /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(6931..7211)
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     primer_bind     complement(7228..7247)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7347..7369
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
       61 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
      121 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
      181 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
      241 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
      301 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
      361 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
      421 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
      481 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
      541 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
      601 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
      661 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
      721 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
      781 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
      841 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
      901 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
      961 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     1021 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     1081 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     1141 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     1201 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     1261 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     1321 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc
     1381 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     1441 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     1501 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     1561 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     1621 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     1681 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     1741 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     1801 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     1861 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     1921 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     1981 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
     2041 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
     2101 acgcaattaa tgtgagttac ctcactcatt aggcacccca ggctttacac tttatgcttc
     2161 cggctcctat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg
     2221 accatgatta cgccaagcgc gcaattaacc ctcactaaag ggaacaaaag ctggagctcc
     2281 agtatagcga ccagcattca catacgattg acgcatgata ttactttctg cgcacttaac
     2341 ttcgcatctg ggcagatgat gtcgaggcga aaaaaaatat aaatcacgct aacatttgat
     2401 taaaatagaa caactacaat ataaaaaaac tatacaaatg acaagttctt gaaaacaaga
     2461 atctttttat tgtcagtact gattagaaaa acctaggggc agggcatgct catgtagagc
     2521 gcctgccgct cgccgtccga ggcggtgccg tcgtacaggg cggtgtccag gccgcagagg
     2581 gtgaacccca tccgccggta cgcgtggatc gccggtgcgt tgacgttggt gacctccagc
     2641 cagaggtgcc cggcgccccg ctcgccggcg aactccgtcg cgagccccat caacgcgcgc
     2701 ccgaccccgt gcccccggtg ctccggggcg acctcgatgt cctcgacggt cagccggcgg
     2761 ttccacgccg agtacgagat gaccacgaag cccgccaggt cgccgtcgtc cccgtacgcg
     2821 acgaacgtcc gggagtccgg gtcgccgtcc tccccgtcgt ccgattcgtc gtccgattcg
     2881 tcgtcgggga acaccttggt caggggcggg tccaccggca cctcccgcag ggtgaagccg
     2941 tccccggtgg cggtgacgcg gaagacggtg tcggtggtga aggacccatc cagtgcctcg
     3001 atggcctcgg cgtcccccgg gacactggtg cggtaccggt aagccgtgtc gtcaagagtg
     3061 gtcatggttg tttatgttcg gatgtgatgt gagaactgta tcctagcaag attttaaaag
     3121 gaagtatatg aaagaagaac ctcagtggca aatcctaacc ttttatattt ctctacaggg
     3181 gcgcggcgtg gggacaattc aacgcgtctg tgaggggagc gtttccctgc tcgcaggtct
     3241 gcagcgagga gccgtaattt ttgcttcgcg ccgtgcggcc atcaaaatgt atggatgcaa
     3301 atgattatac atggggatgt atgggctaaa tgtacgggcg acagtcacat catgcccctg
     3361 agctgcgcac gtcaagactg tcaaggaggg tattctgggc ctccatgtcg agctcagttt
     3421 atcattatca atactcgcca tttcaaagaa tacgtaaata attaatagta gtgattttcc
     3481 taactttatt tagtcaaaaa attagccttt taattctgct gtaacccgta catgcccaaa
     3541 atagggggcg ggttacacag aatatataac atcgtaggtg tctgggtgaa cagtttattc
     3601 ctggcatcca ctaaatataa tggagcccgc tttttaagct ggcatccaga aaaaaaaaga
     3661 atcccagcac caaaatattg ttttcttcac caaccatcag ttcataggtc cattctctta
     3721 gcgcaactac agagaacagg ggcacaaaca ggcaaaaaac gggcacaacc tcaatggagt
     3781 gatgcaacct gcctggagta aatgatgaca caaggcaatt gacccacgca tgtatctatc
     3841 tcattttctt acaccttcta ttaccttctg ctctctctga tttggaaaaa gctgaaaaaa
     3901 aaggttgaaa ccagttccct gaaattattc ccctacttga ctaataagta tataaagacg
     3961 gtaggtattg attgtaattc tgtaaatcta tttcttaaac ttcttaaatt ctacttttat
     4021 agttagtctt ttttttagtt ttaaaacacc agaacttagt ttcgacggat tctagaacta
     4081 gtatgtttct cccaaaattc aatcccataa ggtcattttc ccccatcctc cgggctaaga
     4141 ctcttcttcg ttaccaaaat cggatgtatt tgagcacaga gataagaaaa gctattgaag
     4201 atgccatcga atcggctcca gtggttcttt tcatgaaagg tactcctgaa tttcccaagt
     4261 gtggattttc aagagcaacc attggattat taggaaatca aggcgttgac ccggccaaat
     4321 ttgcggctta taatgtttta gaagacccag agctacgtga aggtatcaaa gagttttcag
     4381 aatggccaac tattccacag ttatatgtaa acaaagaatt cattggtgga tgtgatgtta
     4441 ttacaagtat ggcacgctct ggtgaattgg ccgatttgct agaagaggca caggcattgg
     4501 tacctgaaga agaagaagaa accaaagatc gtggatccta cccatacgat gttccagatt
     4561 acgctgaatt cggtggtggt tctggcggag gttcaatgtc taaaggtgaa gaattgttta
     4621 ctggtgttgt tccaattttg gttgaattgg atggtgatgt taatggtcat aaattttctg
     4681 ttagaggtga aggtgaaggt gatgctacta ttggtaaatt gactttgaaa tttatttgta
     4741 ctactggtaa attgccagtt ccatggccaa ctttggttac tactttgact tatggtgttc
     4801 aatgtttttc tagatatcca gatcatatga aacaacatga tttttttaaa tctgctatgc
     4861 cagaaggtta tgttcaagaa agaactattt cttttaaaga tgatggtaaa tataaaacta
     4921 gagctgttgt taaatttgaa ggtgatactt tggttaatag aattgaattg aaaggtactg
     4981 attttaaaga agatggtaat attttgggtc ataaattgga atataatttt aattctcata
     5041 atgtttatat tactgctgat aaacaaaaaa atggtattaa agctaatttt actgttagac
     5101 ataatgttga agatggttct gttcaattgg ctgatcatta tcaacaaaat actccaattg
     5161 gtgatggtcc agttttgttg ccagataatc attatttgtc tactcaaact gttttgtcta
     5221 aagatccaaa tgaaaaataa ctcgagtcat gtaattagtt atgtcacgct tacattcacg
     5281 ccctcccccc acatccgctc taaccgaaaa ggaaggagtt agacaacctg aagtctaggt
     5341 ccctatttat ttttttatag ttatgttagt attaagaacg ttatttatat ttcaaatttt
     5401 tctttttttt ctgtacagac gcgtgtacgc atgtaacatt atactgaaaa ccttgcttga
     5461 gaaggttttg ggacgctcga aggctttaat ttgcggccgg tacccaattc gccctatagt
     5521 gagtcgtatt acgcgcgctc actggccgtc gttttacaac gtcgtgactg ggaaaaccct
     5581 ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc
     5641 gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatggcgc
     5701 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     5761 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     5821 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     5881 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
     5941 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     6001 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
     6061 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
     6121 aacgcgaatt ttaacaaaat attaacgttt acaatttcct gatgcggtat tttctcctta
     6181 cgcatctgtg cggtatttca caccgcatag gcaagtgcac aaacaatact taaataaata
     6241 ctactcagta ataacctatt tcttagcatt tttgacgaaa tttgctattt tgttagagtc
     6301 ttttacacca tttgtctcca cacctccgct tacatcaaca ccaataacgc catttaatct
     6361 aagcgcatca ccaacatttt ctggcgtcag tccaccagct aacataaaat gtaagctttc
     6421 ggggctctct tgccttccaa cccagtcaga aatcgagttc caatccaaaa gttcacctgt
     6481 cccacctgct tctgaatcaa acaagggaat aaacgaatga ggtttctgtg aagctgcact
     6541 gagtagtatg ttgcagtctt ttggaaatac gagtctttta ataactggca aaccgaggaa
     6601 ctcttggtat tcttgccacg actcatctcc atgcagttgg acgatatcaa tgccgtaatc
     6661 attgaccaga gccaaaacat cctccttagg ttgattacga aacacgccaa ccaagtattt
     6721 cggagtgcct gaactatttt tatatgcttt tacaagactt gaaattttcc ttgcaataac
     6781 cgggtcaatt gttctctttc tattgggcac acatataata cccagcaagt cagcatcgga
     6841 atctagagca cattctgcgg cctctgtgct ctgcaagccg caaactttca ccaatggacc
     6901 agaactacct gtgaaattaa taacagacat actccaagct gcctttgtgt gcttaatcac
     6961 gtatactcac gtgctcaata gtcaccaatg ccctccctct tggccctctc cttttctttt
     7021 ttcgaccgaa ttaattctta atcggcaaaa aaagaaaagc tccggatcaa gattgtacgt
     7081 aaggtgacaa gctatttttc aataaagaat atcttccact actgccatct ggcgtcataa
     7141 ctgcaaagta cacatatatt acgatgctgt ctattaaatg cttcctatat tatatatata
     7201 gtaatgtcgt ttatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     7261 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     7321 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     7381 atcaccgaaa cgcgcga
//
