LOCUS       sequence-200461-        6101 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mitophagy cargo assay.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6101)
  AUTHORS   Dennis PB, Mercer CA
  TITLE     The GST-BHMT assay and related assays for autophagy.
  JOURNAL   Methods Enzymol. 2009;452:97-118. doi: 
            10.1016/S0076-6879(08)03607-0.
  PUBMED    19200878
REFERENCE   2  (bases 1 to 6101)
  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: "Methods 
            Enzymol."; date: "2009"; volume: "452:97-118. doi"; pages: " 
            10.1016/S0076-6879(08)03607-0"
COMMENT     Sequence Label: GST(LSCS)GFP-ActA
FEATURES             Location/Qualifiers
     source          1..6101
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        31..222
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     179..199
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     polyA_signal    291..425
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(315..334)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     369..388
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     regulatory      429..438
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(join(533..1243,1244..1246))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             complement(join(533..1243,1244..1246))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(575..596)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     924..943
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1183..1204
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(1262..1915)
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     complement(1282..1304)
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     primer_bind     complement(1961..1980)
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        complement(1993..2011)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1994..2011)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2221..2245)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        complement(2242..2445)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(2271..2291)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        2446..2825
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     complement(2854..2870)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2854..2870)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2867..2889)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2878..2894
                     /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)."
     promoter        complement(join(2902..2908,2909..2926,2927..2932))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2947..2968
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3085..3102)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3256..3844)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3336..3355)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4015..4806,4807..4875))
                     /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     4638..4657
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4876..4980)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5048..5066
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5104..5126)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5226..5245
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(5262..5717)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5399..5420)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5611..5630
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5915..5937
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5929..5946
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5930..5946
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        complement(join(5966..6101,1..222))
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      join(5980..6101,1..14)
                     /label=SV40 ori
                     /note="SV40 origin of replication"
ORIGIN
        1 gttaggggcg ggatgggcgg agttaggggc gggactatgg ttgctgacta attgagatgc
       61 atgctttgca tacttctgcc tgctggggag cctggggact ttccacacct ggttgctgac
      121 taattgagat gcatgctttg catacttctg cctgctgggg agcctgggga ctttccacac
      181 cctaactgac acacattcca cagctggttc tttccgcctc agaaggtacc taaccaagtt
      241 cctctttcag aggttatttc aggccatggt gctgcgccga attaattccc gatccagaca
      301 tgataagata cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct
      361 ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac
      421 aagttgggcc atggcggcca agcttctagt tgttcttgcg cagctggatg atcttgatga
      481 aggcgcccag gctgaacacg ccgatggcca gcatggccag gatcagtcta gacttgtaca
      541 gctcgtccat gccgagagtg atcccggcgg cggtcacgaa ctccagcagg accatgtgat
      601 cgcgcttctc gttggggtct ttgctcaggg cggactgggt gctcaggtag tggttgtcgg
      661 gcagcagcac ggggccgtcg ccgatggggg tgttctgctg gtagtggtcg gcgagctgca
      721 cgctgccgtc ctcgatgttg tggcggatct tgaagttcac cttgatgccg ttcttctgct
      781 tgtcggccat gatatagacg ttgtggctgt tgtagttgta ctccagcttg tgccccagga
      841 tgttgccgtc ctccttgaag tcgatgccct tcagctcgat gcggttcacc agggtgtcgc
      901 cctcgaactt cacctcggcg cgggtcttgt agttgccgtc gtccttgaag aagatggtgc
      961 gctcctggac gtagccttcg ggcatggcgg acttgaagaa gtcgtgctgc ttcatgtggt
     1021 cggggtagcg gctgaagcac tgcacgccgt aggtcagggt ggtcacgagg gtgggccagg
     1081 gcacgggcag cttgccggtg gtgcagatga acttcagggt cagcttgccg taggtggcat
     1141 cgccctcgcc ctcgccggac acgctgaact tgtggccgtt tacgtcgccg tccagctcga
     1201 ccaggatggg caccaccccg gtgaacagct cctcgccctt gctcacgcgg ccgccatccg
     1261 attttggagg atggtcgcca ccaccaaacg tggcttgcca gccctgcaaa ggccatgcta
     1321 tatacttgct ggatttcaag tacttatcaa tttgtgggat agcttcaata cgttttttaa
     1381 aacaaactaa ttttgggaac gcatccaggc acattgggtc catgtataaa acaacatcaa
     1441 gagcgtcata caacatgaag tcaggatggg ttacatgatc accatttaaa tatgttttat
     1501 gacataaacg atcttcgaac attttcagca tttcaggtag cttgctaaga aaatcaactt
     1561 tgagagtttc aaagtcttta ctatatgcaa ttctcgaaac accgtatcta atatccaaaa
     1621 ccgctccttc aagcattgaa atctctgcac gctcttttgg acaaccaccc aacatgttgt
     1681 gcttgtcagc tatataacgt atgatggcca tagactgtgt taatttaaca tcaccatcaa
     1741 tataataagg aagattggga aactccaaac ccaattcaaa ctttttgttt cgccatttat
     1801 caccttcatc gcgctcatac aaatgctctt catatttttc ttcaagatat tccaaaagaa
     1861 gtcgagtggg ttgcacaagg cccttaattt tccaataacc tagtataggg gacatggctg
     1921 gctcgataga accgaggtgc agttggacct gggagtggac acctgtggag agaaaggcaa
     1981 agtggatgtt attctatagt gtcacctaaa tcgtatgtgt atgatacata aggttatgta
     2041 ttaattgtag ccgcgttcta acgaagccaa gggggtgggc ctatagactc tataggcggt
     2101 acttacgtca ctcttggcac ggggaatccg cgttccaatg caccgttccc ggccgcggag
     2161 gctggatcgg tcccggtgtc ttctatggag gtcaaaacag cgtggatggc gtctccaggc
     2221 gatctgacgg ttcactaaac gagctctgct tatatagacc tcccaccgta cacgcctacc
     2281 gcccatttgc gtcaatgggg cggagttgtt acgacatttt ggaaagtccc gttgattttg
     2341 gtgccaaaac aaactcccat tgacgtcaat ggggtggaga cttggaaatc cccgtgagtc
     2401 aaaccgctat ccacgcccat tgatgtactg ccaaaaccgc atcaccatgg taatagcgat
     2461 gactaatacg tagatgtact gccaagtagg aaagtcccat aaggtcatgt actgggcata
     2521 atgccaggcg ggccatttac cgtcattgac gtcaataggg ggcgtacttg gcatatgata
     2581 cacttgatgt actgccaagt gggcagttta ccgtaaatac tccacccatt gacgtcaatg
     2641 gaaagtccct attggcgtta ctatgggaac atacgtcatt attgacgtca atgggcgggg
     2701 gtcgttgggc ggtcagccag gcgggccatt taccgtaagt tatgtaacgc ggaactccat
     2761 atatgggcta tgaactaatg accccgtaat tgattactat taataactag tcaataatca
     2821 atgtcgggcg agctcgaatt aattcgtaat catgtcatag ctgtttcctg tgtgaaattg
     2881 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg
     2941 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
     3001 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     3061 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     3121 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     3181 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     3241 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     3301 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     3361 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     3421 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     3481 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     3541 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     3601 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     3661 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     3721 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     3781 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     3841 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     3901 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     3961 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     4021 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     4081 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     4141 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     4201 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     4261 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     4321 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     4381 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     4441 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     4501 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     4561 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     4621 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     4681 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     4741 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     4801 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     4861 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     4921 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     4981 cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac
     5041 ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga
     5101 aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg
     5161 gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa
     5221 ctatgcggca tcagagcaga ttgtactgag agtgcaccat aaaattgtaa acgttaatat
     5281 tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga
     5341 aatcggcaaa atcccttata aatcaaaaga atagcccgag atagggttga gtgttgttcc
     5401 agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac
     5461 cgtctatcag ggcgatggcc cactacgtga accatcaccc aaatcaagtt ttttggggtc
     5521 gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg
     5581 gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag
     5641 ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc
     5701 gccgctacag ggcgcgtact atggttgctt tgacgtatgc ggtgtgaaat accgcacaga
     5761 tgcgtaagga gaaaataccg catcaggcgc cattcgccat tcaggctgcg caactgttgg
     5821 gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct
     5881 gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg taaaacgacg
     5941 gccagtgcca agctgttaac agctttttgc aaaagcctag gcctccaaaa aagcctcctc
     6001 actacttctg gaatagctca gaggccgagg cggcctcggc ctctgcataa ataaaaaaaa
     6061 ttagtcagcc atggggcgga gaatgggcgg aactgggcgg a
//
