LOCUS       sequence-197984-        6095 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 6095)
  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 6095)
  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..6095
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        14..393
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        394..597
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     548..568
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     594..618
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        828..846
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     828..845
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     859..878
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     CDS             918..1571
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRAKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     1529..1551
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             join(1587..1589,1590..2300)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(1587..1589,1590..2300)
                     /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(1629..1650)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1890..1909)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2237..2258
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     regulatory      2395..2404
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2408..2542
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2445..2464)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2499..2518
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        2611..2968
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2611..2802
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2634..2654)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2819..2954
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2988..3005)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2988..3004)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2997..3019)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3217..3672
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3304..3323)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3514..3535
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3689..3708)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3808..3830
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3868..3886)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3954..4058
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4059..4127,4128..4919)
                     /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(4277..4296)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5090..5678
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5579..5598
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5832..5849
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5966..5987
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6002..6007,6008..6025,6026..6032)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6040..6056
                     /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     6045..6067
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6064..6080
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6064..6080
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 ttcgagctcg cccgacattg attattgact agttattaat agtaatcaat tacggggtca
       61 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
      121 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
      181 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
      241 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
      301 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
      361 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat
      421 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
      481 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc
      541 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctcgt
      601 ttagtgaacc gtcagatcgc ctggagacgc catccacgct gttttgacct ccatagaaga
      661 caccgggacc gatccagcct ccgcggccgg gaacggtgca ttggaacgcg gattccccgt
      721 gccaagagtg acgtaagtac cgcctataga gtctataggc ccaccccctt ggcttcgtta
      781 gaacgcggct acaattaata cataacctta tgtatcatac acatacgatt taggtgacac
      841 tatagaataa catccacttt gcctttctct ccacaggtgt ccacttccca ggtccaactg
      901 tacctcgcga gccagccatg tcccctatac taggttattg gaaaattaag ggccttgtgc
      961 aacccactcg acttcttttg gaatatcttg aagaaaaata tgaagagcat ttgtatgagc
     1021 gcgatgaagg tgataaatgg cgagccaaaa agtttgaatt gggtttggag tttcccaatc
     1081 ttccttatta tattgatggt gatgttaaat taacacagtc tatggccatc atacgttata
     1141 tagctgacaa gcacaacatg ttgggtggtt gtccaaaaga gcgtgcagag atttcaatgc
     1201 ttgaaggagc ggttttggat attagatacg gtgtttcgag aattgcatat agtaaagact
     1261 ttgaaactct caaagttgat tttcttagca agctacctga aatgctgaaa atgttcgaag
     1321 atcgtttatg tcataaaaca tatttaaatg gtgatcatgt aacccatcct gacttcatgt
     1381 tgtatgacgc tcttgatgtt gttttataca tggacccaat gtgcctggat gcgttcccaa
     1441 aattagtttg ttttaaaaaa cgtattgaag ctatcccaca aattgataag tacttgaaat
     1501 ccagcaagta tatagcatgg cctttgcagg gctggcaagc cacgtttggt ggtggcgacc
     1561 atcctccaaa atcggatggc ggccgcgtga gcaagggcga ggagctgttc accggggtgg
     1621 tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg
     1681 agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca
     1741 agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca
     1801 gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct
     1861 acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg
     1921 tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg
     1981 aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata
     2041 tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg
     2101 aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc
     2161 ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca
     2221 acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg
     2281 gcatggacga gctgtacaag tctagactga tcctggccat gctggccatc ggcgtgttca
     2341 gcctgggcgc cttcatcaag atcatccagc tgcgcaagaa caactagaag cttggccgcc
     2401 atggcccaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
     2461 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
     2521 tgtatcttat catgtctgga tcgggaatta attcggcgca gcaccatggc ctgaaataac
     2581 ctctgaaaga ggaacttggt taggtacctt ctgaggcgga aagaaccagc tgtggaatgt
     2641 gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
     2701 gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag
     2761 tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat
     2821 cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt
     2881 tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg
     2941 cttttttgga ggcctaggct tttgcaaaaa gctgttaaca gcttggcact ggccgtcgtt
     3001 ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat
     3061 ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag
     3121 ttgcgcagcc tgaatggcga atggcgcctg atgcggtatt ttctccttac gcatctgtgc
     3181 ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa
     3241 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     3301 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     3361 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     3421 aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     3481 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     3541 cactcaaccc tatctcgggc tattcttttg atttataagg gattttgccg atttcggcct
     3601 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     3661 cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     3721 agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     3781 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     3841 catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg
     3901 tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa
     3961 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     4021 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     4081 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     4141 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     4201 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     4261 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     4321 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     4381 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     4441 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     4501 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     4561 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     4621 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     4681 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     4741 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     4801 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     4861 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     4921 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     4981 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     5041 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     5101 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     5161 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     5221 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     5281 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     5341 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     5401 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     5461 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     5521 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     5581 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     5641 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     5701 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     5761 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     5821 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc
     5881 ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga
     5941 aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg
     6001 ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc
     6061 acacaggaaa cagctatgac atgattacga attaa
//
