LOCUS       sequence-197983-        6050 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 6050)
  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 6050)
  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..6050
                     /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"
     CDS             2310..2339
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     regulatory      2350..2359
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2363..2497
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2400..2419)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2454..2473
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        2566..2923
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2566..2757
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2589..2609)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2774..2909
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2943..2960)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2943..2959)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2952..2974)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3172..3627
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3259..3278)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3469..3490
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3644..3663)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3763..3785
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3823..3841)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3909..4013
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4014..4082,4083..4874)
                     /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(4232..4251)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5045..5633
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5534..5553
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5787..5804
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5921..5942
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5957..5962,5963..5980,5981..5987)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5995..6011
                     /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     6000..6022
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6019..6035
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6019..6035
                     /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 tctagaatgg agcagaagct gatctccgag gaggacctgt
     2341 gaaagcttgg ccgccatggc ccaacttgtt tattgcagct tataatggtt acaaataaag
     2401 caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt
     2461 gtccaaactc atcaatgtat cttatcatgt ctggatcggg aattaattcg gcgcagcacc
     2521 atggcctgaa ataacctctg aaagaggaac ttggttaggt accttctgag gcggaaagaa
     2581 ccagctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag
     2641 aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc
     2701 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc
     2761 cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg
     2821 ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca
     2881 gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagctgt taacagcttg
     2941 gcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat
     3001 cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat
     3061 cgcccttccc aacagttgcg cagcctgaat ggcgaatggc gcctgatgcg gtattttctc
     3121 cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct
     3181 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     3241 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     3301 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     3361 ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg ccatcgccct
     3421 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     3481 tccaaactgg aacaacactc aaccctatct cgggctattc ttttgattta taagggattt
     3541 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     3601 ttaacaaaat attaacgttt acaattttat ggtgcactct cagtacaatc tgctctgatg
     3661 ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt
     3721 gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc
     3781 agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat
     3841 ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg
     3901 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     3961 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     4021 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     4081 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     4141 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     4201 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     4261 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     4321 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     4381 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     4441 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     4501 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     4561 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     4621 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     4681 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     4741 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     4801 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     4861 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     4921 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     4981 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     5041 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     5101 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     5161 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
     5221 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     5281 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     5341 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     5401 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     5461 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     5521 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     5581 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     5641 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     5701 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     5761 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
     5821 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca
     5881 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc
     5941 attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga
     6001 gcggataaca atttcacaca ggaaacagct atgacatgat tacgaattaa
//
