LOCUS       Exported                7214 bp ds-DNA     circular SYN 20-JAN-2022
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    p406 – TDH3-ER-FlucDM-GFP11-Ura
            
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7214)
  TITLE     ER-misfolded proteins become sequestered with mitochondria and 
            impair mitochondrial function
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7214)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jan 20, 2022 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7214
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     complement(19..37)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        105..209
                     /gene="bla"
                     /label=AmpR promoter
     CDS             210..1070
                     /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(428..447)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1241..1829
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1730..1749
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1983..2000
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2117..2138
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        2153..2183
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2191..2207
                     /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     2196..2218
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2215..2231
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2215..2231
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     2250..2270
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2252..2270
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        2295..2938
                     /gene="S. cerevisiae TDH3"
                     /label=GAP promoter
                     /note="promoter for glyceraldehyde-3-phosphate 
                     dehydrogenase"
     primer_bind     2832..2855
                     /label=GPDpro-F
                     /note="S. cerevisiae GPD promoter, forward primer"
     CDS             3124..4770
                     /codon_start=1
                     /product="firefly luciferase"
                     /label=luciferase
                     /note="synthetic luc2 version of the luciferase gene"
                     /translation="EDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDAH
                     IEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAPA
                     NDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQSM
                     YTFVTSHLPPGFNEYDFVPESFDQDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHAR
                     DPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFQVVLMYRFEEELFLRSLQDYKI
                     QSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGYG
                     LTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMSG
                     YVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESILL
                     QHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVVF
                     VDEVPKGLTGKLDARKIREILIKAKKGGKIAV"
     CDS             4807..4833
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             4873..4920
                     /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(4955..4971)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4956..4972)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     terminator      4974..5221
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(4990..5008)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(5239..5258)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5240..5258)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5268..5285)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5268..5284)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5277..5299)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5425..5880
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5512..5531)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5722..5743
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(6011..6814)
                     /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(6815..7030)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(7054..7073)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7173..7195
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa
       61 taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
      121 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
      181 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
      241 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
      301 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
      361 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
      421 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
      481 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
      541 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
      601 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
      661 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
      721 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
      781 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
      841 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
      901 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
      961 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     1021 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     1081 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     1141 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     1201 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     1261 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     1321 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     1381 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     1441 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     1501 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     1561 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     1621 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     1681 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     1741 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     1801 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     1861 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     1921 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     1981 gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg
     2041 cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca
     2101 gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact
     2161 ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa
     2221 acagctatga ccatgattac gccaagcgcg caattaaccc tcactaaagg gaacaaaagc
     2281 tggagctcag tttatcatta tcaatactcg ccatttcaaa gaatacgtaa ataattaata
     2341 gtagtgattt tcctaacttt atttagtcaa aaaattagcc ttttaattct gctgtaaccc
     2401 gtacatgccc aaaatagggg gcgggttaca cagaatatat aacatcgtag gtgtctgggt
     2461 gaacagttta ttcctggcat ccactaaata taatggagcc cgctttttaa gctggcatcc
     2521 agaaaaaaaa agaatcccag caccaaaata ttgttttctt caccaaccat cagttcatag
     2581 gtccattctc ttagcgcaac tacagagaac aggggcacaa acaggcaaaa aacgggcaca
     2641 acctcaatgg agtgatgcaa cctgcctgga gtaaatgatg acacaaggca attgacccac
     2701 gcatgtatct atctcatttt cttacacctt ctattacctt ctgctctctc tgatttggaa
     2761 aaagctgaaa aaaaaggttg aaaccagttc cctgaaatta ttcccctact tgactaataa
     2821 gtatataaag acggtaggta ttgattgtaa ttctgtaaat ctatttctta aacttcttaa
     2881 attctacttt tatagttagt ctttttttta gttttaaaac accagaactt agtttcgacg
     2941 gattctagaa ctagtatgtt tttcaacaga ctaagcgctg gcaagctgct ggtaccactc
     3001 tccgtggtcc tgtacgccct tttcgtggta atattacctt tacagaattc tttccactcc
     3061 tccaatgttt tagttagagg tttggatcct gacttaggta ctacttattc cttgctagct
     3121 gtggaagatg ccaaaaacat taagaagggc ccagcgccat tctacccact cgaagacggg
     3181 accgccggcg agcagctgca caaagccatg aagcgctacg ccctggtgcc cggcaccatc
     3241 gcctttaccg acgcacatat cgaggtggac attacctacg ccgagtactt cgagatgagc
     3301 gttcggctgg cagaagctat gaagcgctat gggctgaata caaaccatcg gatcgtggtg
     3361 tgcagcgaga atagcttgca gttcttcatg cccgtgttgg gtgccctgtt catcggtgtg
     3421 gctgtggccc cagctaacga catctacaac gagcgcgagc tgctgaacag catgggcatc
     3481 agccagccca ccgtcgtatt cgtgagcaag aaagggctgc aaaagatcct caacgtgcaa
     3541 aagaagctac cgatcataca aaagatcatc atcatggata gcaagaccga ctaccagggc
     3601 ttccaaagca tgtacacctt cgtgacttcc catttgccac ccggcttcaa cgagtacgac
     3661 ttcgtgcccg agagcttcga ccaggacaaa accatcgccc tgatcatgaa cagtagtggc
     3721 agtaccggat tgcccaaggg cgtagcccta ccgcaccgca ccgcttgtgt ccgattcagt
     3781 catgcccgcg accccatctt cggcaaccag atcatccccg acaccgctat cctcagcgtg
     3841 gtgccatttc accacggctt cggcatgttc accacgctgg gctacttgat ctgcggcttt
     3901 caagtcgtgc tcatgtaccg cttcgaggag gagctattct tgcgcagctt gcaagactat
     3961 aagattcaat ctgccctgct ggtgcccaca ctatttagct tcttcgctaa gagcactctc
     4021 atcgacaagt acgacctaag caacttgcac gagatcgcca gcggcggggc gccgctcagc
     4081 aaggaggtag gtgaggccgt ggccaaacgc ttccacctac caggcatccg ccagggctac
     4141 ggcctgacag aaacaaccag cgccattctg atcacccccg aaggggacga caagcctggc
     4201 gcagtaggca aggtggtgcc cttcttcgag gctaaggtgg tggacttgga caccggtaag
     4261 acactgggtg tgaaccagcg cggcgagctg tgcgtccgtg gccccatgat catgagcggc
     4321 tacgttaaca accccgaggc tacaaacgct ctcatcgaca aggacggctg gctgcacagc
     4381 ggcgacatcg cctactggga cgaggacgag cacttcttca tcgtggaccg gctgaagagc
     4441 ctgatcaaat acaagggcta ccaggtagcc ccagccgaac tggagagcat cctgctgcaa
     4501 caccccaaca tcttcgacgc cggggtcgcc ggcctgcccg acgacgatgc cggcgagctg
     4561 cccgccgcag tcgtcgtgct ggaacacggt aaaaccatga ccgagaagga gatcgtggac
     4621 tatgtggcca gccaggttac aaccgccaag aagctgcgcg gtggtgttgt gttcgtggac
     4681 gaggtgccta aaggactgac cggcaagttg gacgcccgca agatccgcga gattctcatt
     4741 aaggccaaga agggcggcaa gatcgccgtg atccccgggg gatcgggtga cggtccggtc
     4801 gccacctacc catacgatgt tccagattac gctggtcgac ggatcggtgg tggttctgga
     4861 ggcggttcta tgagagatca tatggttttg catgaatatg ttaatgctgc tggtattact
     4921 aaaaagaccc acgacgaatt gtagtaaagc ttatcgatac cgtcgacctc gagtcatgta
     4981 attagttatg tcacgcttac attcacgccc tccccccaca tccgctctaa ccgaaaagga
     5041 aggagttaga caacctgaag tctaggtccc tatttatttt tttatagtta tgttagtatt
     5101 aagaacgtta tttatatttc aaatttttct tttttttctg tacagacgcg tgtacgcatg
     5161 taacattata ctgaaaacct tgcttgagaa ggttttggga cgctcgaagg ctttaatttg
     5221 cggccggtac ccaattcgcc ctatagtgag tcgtattacg cgcgctcact ggccgtcgtt
     5281 ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat
     5341 ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag
     5401 ttgcgcagcc tgaatggcga atggacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     5461 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     5521 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     5581 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     5641 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     5701 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     5761 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     5821 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt
     5881 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tagggtaata
     5941 actgatataa ttaaattgaa gctctaattt gtgagtttag tatacatgca tttacttata
     6001 atacagtttt ttagttttgc tggccgcatc ttctcaaata tgcttcccag cctgcttttc
     6061 tgtaacgttc accctctacc ttagcatccc ttccctttgc aaatagtcct cttccaacaa
     6121 taataatgtc agatcctgta gagaccacat catccacggt tctatactgt tgacccaatg
     6181 cgtctccctt gtcatctaaa cccacaccgg gtgtcataat caaccaatcg taaccttcat
     6241 ctcttccacc catgtctctt tgagcaataa agccgataac aaaatctttg tcgctcttcg
     6301 caatgtcaac agtaccctta gtatattctc cagtagatag ggagcccttg catgacaatt
     6361 ctgctaacat caaaaggcct ctaggttcct ttgttacttc ttctgccgcc tgcttcaaac
     6421 cgctaacaat acctgggccc accacaccgt gtgcattcgt aatgtctgcc cattctgcta
     6481 ttctgtatac acccgcagag tactgcaatt tgactgtatt accaatgtca gcaaattttc
     6541 tgtcttcgaa gagtaaaaaa ttgtacttgg cggataatgc ctttagcggc ttaactgtgc
     6601 cctccatgga aaaatcagtc aagatatcca catgtgtttt tagtaaacaa attttgggac
     6661 ctaatgcttc aactaactcc agtaattcct tggtggtacg aacatccaat gaagcacaca
     6721 agtttgtttg cttttcgtgc atgatattaa atagcttggc agcaacagga ctaggatgag
     6781 tagcagcacg ttccttatat gtagctttcg acatgattta tcttcgtttc ctgcaggttt
     6841 ttgttctgtg cagttgggtt aagaatactg ggcaatttca tgtttcttca acactacata
     6901 tgcgtatata taccaatcta agtctgtgct ccttccttcg ttcttccttc tgttcggaga
     6961 ttaccgaatc aaaaaaattt caaggaaacc gaaatcaaaa aaaagaataa aaaaaaaatg
     7021 atgaattgaa aaggtggtat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     7081 aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     7141 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     7201 accgtcatca ccga
//
