LOCUS       sequence-344864-        7228 bp    DNA     circular SYN 15-APR-2025
DEFINITION  DM Firefly luciferase reporter fused to a KAR2 signal sequence for 
            ER targeting and HDEL for ER retention with an HA tag and the 
            eleventh beta-strand of GFP added to the C-terminus in yeast plasmid
            with.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7228)
  AUTHORS   Cortes Sanchon A, Santhosh Kumar H, Mantovani M, Osinnii I, Mateos 
            JM, Kaech A, Shcherbakov D, Akbergenov R, Bottger EC
  TITLE     ER-misfolded proteins become sequestered with mitochondria and 
            impair mitochondrial function.
  JOURNAL   Commun Biol. 2021 Dec 2;4(1):1350. doi: 10.1038/s42003-021-02873-w.
  PUBMED    34857875
REFERENCE   2  (bases 1 to 7228)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Commun 
            Biol."; date: "2021-12-2"; volume: "4(1):1350. doi"; pages: " 
            10.1038/s42003-021-02873-w"
COMMENT     Sequence Label: p406 – TDH3-ER-FlucDM-GFP11-Ura
            
FEATURES             Location/Qualifiers
     source          1..7228
                     /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"
     promoter        2286..2929
                     /gene="S. cerevisiae TDH3"
                     /label=GAP promoter
                     /note="promoter for glyceraldehyde-3-phosphate 
                     dehydrogenase; also known as the TDH3 promoter"
     primer_bind     2823..2846
                     /label=GPDpro-F
                     /note="S. cerevisiae GPD promoter, forward primer"
     CDS             3115..4764
                     /codon_start=1
                     /product="firefly luciferase"
                     /label=luciferase
                     /note="synthetic luc2 version of the luciferase gene"
                     /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA
                     HIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP
                     ANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS
                     MYTFVTSHLPPGFNEYDFVPESFDQDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
                     RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFQVVLMYRFEEELFLRSLQDYK
                     IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY
                     GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS
                     GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL
                     LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV
                     FVDEVPKGLTGKLDARKIREILIKAKKGGKIAV"
     CDS             4801..4827
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             4867..4914
                     /codon_start=1
                     /product="11th beta-strand of superfolder GFP"
                     /label=GFP11
                     /note="associates with GFP(1-10), consisting of the first 
                     10 beta-strands, to reconstitute fluorescent GFP (Cabantous
                     et al., 2005; Kamiyama et al., 2016)"
                     /translation="RDHMVLHEYVNAAGIT"
     primer_bind     complement(4949..4965)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4950..4966)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     terminator      4968..5215
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(4984..5002)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(5233..5252)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5234..5252)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5262..5279)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5262..5278)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5271..5293)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5423..5878
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5510..5529)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5720..5741
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(6009..6812)
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     promoter        complement(6813..7033)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(7059..7078)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7178..7200
                     /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 ctggagctca
     2281 gtttatcatt atcaatactc gccatttcaa agaatacgta aataattaat agtagtgatt
     2341 ttcctaactt tatttagtca aaaaattagc cttttaattc tgctgtaacc cgtacatgcc
     2401 caaaataggg ggcgggttac acagaatata taacatcgta ggtgtctggg tgaacagttt
     2461 attcctggca tccactaaat ataatggagc ccgcttttta agctggcatc cagaaaaaaa
     2521 aagaatccca gcaccaaaat attgttttct tcaccaacca tcagttcata ggtccattct
     2581 cttagcgcaa ctacagagaa caggggcaca aacaggcaaa aaacgggcac aacctcaatg
     2641 gagtgatgca acctgcctgg agtaaatgat gacacaaggc aattgaccca cgcatgtatc
     2701 tatctcattt tcttacacct tctattacct tctgctctct ctgatttgga aaaagctgaa
     2761 aaaaaaggtt gaaaccagtt ccctgaaatt attcccctac ttgactaata agtatataaa
     2821 gacggtaggt attgattgta attctgtaaa tctatttctt aaacttctta aattctactt
     2881 ttatagttag tctttttttt agttttaaaa caccagaact tagtttcgac ggattctaga
     2941 actagtatgt ttttcaacag actaagcgct ggcaagctgc tggtaccact ctccgtggtc
     3001 ctgtacgccc ttttcgtggt aatattacct ttacagaatt ctttccactc ctccaatgtt
     3061 ttagttagag gtttggatcc tgacttaggt actacttatt ccttgctagc tgtgatggaa
     3121 gatgccaaaa acattaagaa gggcccagcg ccattctacc cactcgaaga cgggaccgcc
     3181 ggcgagcagc tgcacaaagc catgaagcgc tacgccctgg tgcccggcac catcgccttt
     3241 accgacgcac atatcgaggt ggacattacc tacgccgagt acttcgagat gagcgttcgg
     3301 ctggcagaag ctatgaagcg ctatgggctg aatacaaacc atcggatcgt ggtgtgcagc
     3361 gagaatagct tgcagttctt catgcccgtg ttgggtgccc tgttcatcgg tgtggctgtg
     3421 gccccagcta acgacatcta caacgagcgc gagctgctga acagcatggg catcagccag
     3481 cccaccgtcg tattcgtgag caagaaaggg ctgcaaaaga tcctcaacgt gcaaaagaag
     3541 ctaccgatca tacaaaagat catcatcatg gatagcaaga ccgactacca gggcttccaa
     3601 agcatgtaca ccttcgtgac ttcccatttg ccacccggct tcaacgagta cgacttcgtg
     3661 cccgagagct tcgaccagga caaaaccatc gccctgatca tgaacagtag tggcagtacc
     3721 ggattgccca agggcgtagc cctaccgcac cgcaccgctt gtgtccgatt cagtcatgcc
     3781 cgcgacccca tcttcggcaa ccagatcatc cccgacaccg ctatcctcag cgtggtgcca
     3841 tttcaccacg gcttcggcat gttcaccacg ctgggctact tgatctgcgg ctttcaagtc
     3901 gtgctcatgt accgcttcga ggaggagcta ttcttgcgca gcttgcaaga ctataagatt
     3961 caatctgccc tgctggtgcc cacactattt agcttcttcg ctaagagcac tctcatcgac
     4021 aagtacgacc taagcaactt gcacgagatc gccagcggcg gggcgccgct cagcaaggag
     4081 gtaggtgagg ccgtggccaa acgcttccac ctaccaggca tccgccaggg ctacggcctg
     4141 acagaaacaa ccagcgccat tctgatcacc cccgaagggg acgacaagcc tggcgcagta
     4201 ggcaaggtgg tgcccttctt cgaggctaag gtggtggact tggacaccgg taagacactg
     4261 ggtgtgaacc agcgcggcga gctgtgcgtc cgtggcccca tgatcatgag cggctacgtt
     4321 aacaaccccg aggctacaaa cgctctcatc gacaaggacg gctggctgca cagcggcgac
     4381 atcgcctact gggacgagga cgagcacttc ttcatcgtgg accggctgaa gagcctgatc
     4441 aaatacaagg gctaccaggt agccccagcc gaactggaga gcatcctgct gcaacacccc
     4501 aacatcttcg acgccggggt cgccggcctg cccgacgacg atgccggcga gctgcccgcc
     4561 gcagtcgtcg tgctggaaca cggtaaaacc atgaccgaga aggagatcgt ggactatgtg
     4621 gccagccagg ttacaaccgc caagaagctg cgcggtggtg ttgtgttcgt ggacgaggtg
     4681 cctaaaggac tgaccggcaa gttggacgcc cgcaagatcc gcgagattct cattaaggcc
     4741 aagaagggcg gcaagatcgc cgtgatcccc gggggatcgg gtgacggtcc ggtcgccacc
     4801 tacccatacg atgttccaga ttacgctggt cgacggatcg gtggtggttc tggaggcggt
     4861 tctatgagag atcatatggt tttgcatgaa tatgttaatg ctgctggtat tactaaaaag
     4921 acccacgacg aattgtagta aagcttatcg ataccgtcga cctcgagtca tgtaattagt
     4981 tatgtcacgc ttacattcac gccctccccc cacatccgct ctaaccgaaa aggaaggagt
     5041 tagacaacct gaagtctagg tccctattta tttttttata gttatgttag tattaagaac
     5101 gttatttata tttcaaattt ttcttttttt tctgtacaga cgcgtgtacg catgtaacat
     5161 tatactgaaa accttgcttg agaaggtttt gggacgctcg aaggctttaa tttgcggccg
     5221 gtacccaatt cgccctatag tgagtcgtat tacgcgcgct cactggccgt cgttttacaa
     5281 cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct
     5341 ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc
     5401 agcctgaatg gcgaatggcg cgacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     5461 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     5521 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     5581 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
     5641 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     5701 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
     5761 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
     5821 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttcc
     5881 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata gggtaataac
     5941 tgatataatt aaattgaagc tctaatttgt gagtttagta tacatgcatt tacttataat
     6001 acagtttttt agttttgctg gccgcatctt ctcaaatatg cttcccagcc tgcttttctg
     6061 taacgttcac cctctacctt agcatccctt ccctttgcaa atagtcctct tccaacaata
     6121 ataatgtcag atcctgtaga gaccacatca tccacggttc tatactgttg acccaatgcg
     6181 tctcccttgt catctaaacc cacaccgggt gtcataatca accaatcgta accttcatct
     6241 cttccaccca tgtctctttg agcaataaag ccgataacaa aatctttgtc gctcttcgca
     6301 atgtcaacag tacccttagt atattctcca gtagataggg agcccttgca tgacaattct
     6361 gctaacatca aaaggcctct aggttccttt gttacttctt ctgccgcctg cttcaaaccg
     6421 ctaacaatac ctgggcccac cacaccgtgt gcattcgtaa tgtctgccca ttctgctatt
     6481 ctgtatacac ccgcagagta ctgcaatttg actgtattac caatgtcagc aaattttctg
     6541 tcttcgaaga gtaaaaaatt gtacttggcg gataatgcct ttagcggctt aactgtgccc
     6601 tccatggaaa aatcagtcaa gatatccaca tgtgttttta gtaaacaaat tttgggacct
     6661 aatgcttcaa ctaactccag taattccttg gtggtacgaa catccaatga agcacacaag
     6721 tttgtttgct tttcgtgcat gatattaaat agcttggcag caacaggact aggatgagta
     6781 gcagcacgtt ccttatatgt agctttcgac atgatttatc ttcgtttcct gcaggttttt
     6841 gttctgtgca gttgggttaa gaatactggg caatttcatg tttcttcaac actacatatg
     6901 cgtatatata ccaatctaag tctgtgctcc ttccttcgtt cttccttctg ttcggagatt
     6961 accgaatcaa aaaaatttca aagaaaccga aatcaaaaaa aagaataaaa aaaaaatgat
     7021 gaattgaatt gaaaagctgt ggtatggtgc actctcagta caatctgctc tgatgccgca
     7081 tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
     7141 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg
     7201 ttttcaccgt catcaccgaa acgcgcga
//
