LOCUS       sequence-200448-        6056 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Autophagy cargo assay.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6056)
  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 6056)
  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)GFPmyc
FEATURES             Location/Qualifiers
     source          1..6056
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        65..256
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     213..233
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     polyA_signal    325..459
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(349..368)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     403..422
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     regulatory      463..472
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(483..512)
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             complement(join(522..1232,1233..1235))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             complement(join(522..1232,1233..1235))
                     /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(564..585)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     913..932
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1172..1193
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(1251..1904)
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     complement(1271..1293)
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     primer_bind     complement(1950..1969)
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        complement(1982..2000)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1983..2000)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2210..2234)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        complement(2231..2434)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(2260..2280)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        2435..2814
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     complement(2843..2859)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2843..2859)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2856..2878)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2867..2883
                     /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(2891..2897,2898..2915,2916..2921))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2936..2957
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3074..3091)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3245..3833)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3325..3344)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4004..4795,4796..4864))
                     /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     4627..4646
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4865..4969)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5037..5055
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5093..5115)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5215..5234
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(5251..5706)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5388..5409)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5600..5619
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5904..5926
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5918..5935
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5919..5935
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        complement(join(5955..6056,1..256))
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      join(5969..6056,1..48)
                     /label=SV40 ori
                     /note="SV40 origin of replication"
ORIGIN
        1 gccatggggc ggagaatggg cggaactggg cggagttagg ggcgggatgg gcggagttag
       61 gggcgggact atggttgctg actaattgag atgcatgctt tgcatacttc tgcctgctgg
      121 ggagcctggg gactttccac acctggttgc tgactaattg agatgcatgc tttgcatact
      181 tctgcctgct ggggagcctg gggactttcc acaccctaac tgacacacat tccacagctg
      241 gttctttccg cctcagaagg tacctaacca agttcctctt tcagaggtta tttcaggcca
      301 tggtgctgcg ccgaattaat tcccgatcca gacatgataa gatacattga tgagtttgga
      361 caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt
      421 gctttatttg taaccattat aagctgcaat aaacaagttg ggccatggcg gccaagcttt
      481 cacaggtcct cctcggagat cagcttctgc tccattctag acttgtacag ctcgtccatg
      541 ccgagagtga tcccggcggc ggtcacgaac tccagcagga ccatgtgatc gcgcttctcg
      601 ttggggtctt tgctcagggc ggactgggtg ctcaggtagt ggttgtcggg cagcagcacg
      661 gggccgtcgc cgatgggggt gttctgctgg tagtggtcgg cgagctgcac gctgccgtcc
      721 tcgatgttgt ggcggatctt gaagttcacc ttgatgccgt tcttctgctt gtcggccatg
      781 atatagacgt tgtggctgtt gtagttgtac tccagcttgt gccccaggat gttgccgtcc
      841 tccttgaagt cgatgccctt cagctcgatg cggttcacca gggtgtcgcc ctcgaacttc
      901 acctcggcgc gggtcttgta gttgccgtcg tccttgaaga agatggtgcg ctcctggacg
      961 tagccttcgg gcatggcgga cttgaagaag tcgtgctgct tcatgtggtc ggggtagcgg
     1021 ctgaagcact gcacgccgta ggtcagggtg gtcacgaggg tgggccaggg cacgggcagc
     1081 ttgccggtgg tgcagatgaa cttcagggtc agcttgccgt aggtggcatc gccctcgccc
     1141 tcgccggaca cgctgaactt gtggccgttt acgtcgccgt ccagctcgac caggatgggc
     1201 accaccccgg tgaacagctc ctcgcccttg ctcacgcggc cgccatccga ttttggagga
     1261 tggtcgccac caccaaacgt ggcttgccag ccctgcaaag gccatgctat atacttgctg
     1321 gatttcaagt acttatcaat ttgtgggata gcttcaatac gttttttaaa acaaactaat
     1381 tttgggaacg catccaggca cattgggtcc atgtataaaa caacatcaag agcgtcatac
     1441 aacatgaagt caggatgggt tacatgatca ccatttaaat atgttttatg acataaacga
     1501 tcttcgaaca ttttcagcat ttcaggtagc ttgctaagaa aatcaacttt gagagtttca
     1561 aagtctttac tatatgcaat tctcgaaaca ccgtatctaa tatccaaaac cgctccttca
     1621 agcattgaaa tctctgcacg ctcttttgga caaccaccca acatgttgtg cttgtcagct
     1681 atataacgta tgatggccat agactgtgtt aatttaacat caccatcaat ataataagga
     1741 agattgggaa actccaaacc caattcaaac tttttgtttc gccatttatc accttcatcg
     1801 cgctcataca aatgctcttc atatttttct tcaagatatt ccaaaagaag tcgagtgggt
     1861 tgcacaaggc ccttaatttt ccaataacct agtatagggg acatggctgg ctcgatagaa
     1921 ccgaggtgca gttggacctg ggagtggaca cctgtggaga gaaaggcaaa gtggatgtta
     1981 ttctatagtg tcacctaaat cgtatgtgta tgatacataa ggttatgtat taattgtagc
     2041 cgcgttctaa cgaagccaag ggggtgggcc tatagactct ataggcggta cttacgtcac
     2101 tcttggcacg gggaatccgc gttccaatgc accgttcccg gccgcggagg ctggatcggt
     2161 cccggtgtct tctatggagg tcaaaacagc gtggatggcg tctccaggcg atctgacggt
     2221 tcactaaacg agctctgctt atatagacct cccaccgtac acgcctaccg cccatttgcg
     2281 tcaatggggc ggagttgtta cgacattttg gaaagtcccg ttgattttgg tgccaaaaca
     2341 aactcccatt gacgtcaatg gggtggagac ttggaaatcc ccgtgagtca aaccgctatc
     2401 cacgcccatt gatgtactgc caaaaccgca tcaccatggt aatagcgatg actaatacgt
     2461 agatgtactg ccaagtagga aagtcccata aggtcatgta ctgggcataa tgccaggcgg
     2521 gccatttacc gtcattgacg tcaatagggg gcgtacttgg catatgatac acttgatgta
     2581 ctgccaagtg ggcagtttac cgtaaatact ccacccattg acgtcaatgg aaagtcccta
     2641 ttggcgttac tatgggaaca tacgtcatta ttgacgtcaa tgggcggggg tcgttgggcg
     2701 gtcagccagg cgggccattt accgtaagtt atgtaacgcg gaactccata tatgggctat
     2761 gaactaatga ccccgtaatt gattactatt aataactagt caataatcaa tgtcgggcga
     2821 gctcgaatta attcgtaatc atgtcatagc tgtttcctgt gtgaaattgt tatccgctca
     2881 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     2941 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     3001 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     3061 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     3121 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     3181 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     3241 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     3301 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     3361 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     3421 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     3481 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     3541 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     3601 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     3661 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     3721 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     3781 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     3841 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     3901 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     3961 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     4021 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     4081 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     4141 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     4201 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     4261 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     4321 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     4381 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     4441 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     4501 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     4561 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     4621 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     4681 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     4741 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     4801 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     4861 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     4921 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     4981 gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc tataaaaata
     5041 ggcgtatcac gaggcccttt cgtctcgcgc gtttcggtga tgacggtgaa aacctctgac
     5101 acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag
     5161 cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac tatgcggcat
     5221 cagagcagat tgtactgaga gtgcaccata aaattgtaaa cgttaatatt ttgttaaaat
     5281 tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa
     5341 tcccttataa atcaaaagaa tagcccgaga tagggttgag tgttgttcca gtttggaaca
     5401 agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg
     5461 gcgatggccc actacgtgaa ccatcaccca aatcaagttt tttggggtcg aggtgccgta
     5521 aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg
     5581 cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa
     5641 gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg
     5701 gcgcgtacta tggttgcttt gacgtatgcg gtgtgaaata ccgcacagat gcgtaaggag
     5761 aaaataccgc atcaggcgcc attcgccatt caggctgcgc aactgttggg aagggcgatc
     5821 ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt
     5881 aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgccaa
     5941 gctgttaaca gctttttgca aaagcctagg cctccaaaaa agcctcctca ctacttctgg
     6001 aatagctcag aggccgaggc ggcctcggcc tctgcataaa taaaaaaaat tagtca
//
