LOCUS       sequence-200480-        6128 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Reticulophagy cargo assay.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6128)
  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 6128)
  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-cb5
FEATURES             Location/Qualifiers
     source          1..6128
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        100..291
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     248..268
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     polyA_signal    360..494
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(384..403)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     438..457
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     regulatory      498..507
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(join(629..1339,1340..1342))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             complement(join(629..1339,1340..1342))
                     /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(671..692)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1020..1039
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1279..1300
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(1358..2011)
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     complement(1378..1400)
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     primer_bind     complement(2057..2076)
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        complement(2089..2107)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2090..2107)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2317..2341)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        complement(2338..2541)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(2367..2387)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        2542..2921
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     complement(2950..2966)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2950..2966)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2963..2985)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2974..2990
                     /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(2998..3004,3005..3022,3023..3028))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3043..3064
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3181..3198)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3352..3940)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3432..3451)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4111..4902,4903..4971))
                     /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     4734..4753
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4972..5076)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5144..5162
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5200..5222)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5322..5341
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(5358..5813)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5495..5516)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5707..5726
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6011..6033
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6025..6042
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6026..6042
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        complement(join(6062..6128,1..291))
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      join(6076..6128,1..83)
                     /label=SV40 ori
                     /note="SV40 origin of replication"
ORIGIN
        1 gcctcggcct ctgcataaat aaaaaaaatt agtcagccat ggggcggaga atgggcggaa
       61 ctgggcggag ttaggggcgg gatgggcgga gttaggggcg ggactatggt tgctgactaa
      121 ttgagatgca tgctttgcat acttctgcct gctggggagc ctggggactt tccacacctg
      181 gttgctgact aattgagatg catgctttgc atacttctgc ctgctgggga gcctggggac
      241 tttccacacc ctaactgaca cacattccac agctggttct ttccgcctca gaaggtacct
      301 aaccaagttc ctctttcaga ggttatttca ggccatggtg ctgcgccgaa ttaattcccg
      361 atccagacat gataagatac attgatgagt ttggacaaac cacaactaga atgcagtgaa
      421 aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct
      481 gcaataaaca agttgggcca tggcggccaa gcttctagtc ctcggccatg tacaggcggt
      541 acatcagggc caccaccagg gcggagatgg cggggatcac ccagttggtc caccaggagg
      601 agttggactc cacggtggtg attctagact tgtacagctc gtccatgccg agagtgatcc
      661 cggcggcggt cacgaactcc agcaggacca tgtgatcgcg cttctcgttg gggtctttgc
      721 tcagggcgga ctgggtgctc aggtagtggt tgtcgggcag cagcacgggg ccgtcgccga
      781 tgggggtgtt ctgctggtag tggtcggcga gctgcacgct gccgtcctcg atgttgtggc
      841 ggatcttgaa gttcaccttg atgccgttct tctgcttgtc ggccatgata tagacgttgt
      901 ggctgttgta gttgtactcc agcttgtgcc ccaggatgtt gccgtcctcc ttgaagtcga
      961 tgcccttcag ctcgatgcgg ttcaccaggg tgtcgccctc gaacttcacc tcggcgcggg
     1021 tcttgtagtt gccgtcgtcc ttgaagaaga tggtgcgctc ctggacgtag ccttcgggca
     1081 tggcggactt gaagaagtcg tgctgcttca tgtggtcggg gtagcggctg aagcactgca
     1141 cgccgtaggt cagggtggtc acgagggtgg gccagggcac gggcagcttg ccggtggtgc
     1201 agatgaactt cagggtcagc ttgccgtagg tggcatcgcc ctcgccctcg ccggacacgc
     1261 tgaacttgtg gccgtttacg tcgccgtcca gctcgaccag gatgggcacc accccggtga
     1321 acagctcctc gcccttgctc acgcggccgc catccgattt tggaggatgg tcgccaccac
     1381 caaacgtggc ttgccagccc tgcaaaggcc atgctatata cttgctggat ttcaagtact
     1441 tatcaatttg tgggatagct tcaatacgtt ttttaaaaca aactaatttt gggaacgcat
     1501 ccaggcacat tgggtccatg tataaaacaa catcaagagc gtcatacaac atgaagtcag
     1561 gatgggttac atgatcacca tttaaatatg ttttatgaca taaacgatct tcgaacattt
     1621 tcagcatttc aggtagcttg ctaagaaaat caactttgag agtttcaaag tctttactat
     1681 atgcaattct cgaaacaccg tatctaatat ccaaaaccgc tccttcaagc attgaaatct
     1741 ctgcacgctc ttttggacaa ccacccaaca tgttgtgctt gtcagctata taacgtatga
     1801 tggccataga ctgtgttaat ttaacatcac catcaatata ataaggaaga ttgggaaact
     1861 ccaaacccaa ttcaaacttt ttgtttcgcc atttatcacc ttcatcgcgc tcatacaaat
     1921 gctcttcata tttttcttca agatattcca aaagaagtcg agtgggttgc acaaggccct
     1981 taattttcca ataacctagt ataggggaca tggctggctc gatagaaccg aggtgcagtt
     2041 ggacctggga gtggacacct gtggagagaa aggcaaagtg gatgttattc tatagtgtca
     2101 cctaaatcgt atgtgtatga tacataaggt tatgtattaa ttgtagccgc gttctaacga
     2161 agccaagggg gtgggcctat agactctata ggcggtactt acgtcactct tggcacgggg
     2221 aatccgcgtt ccaatgcacc gttcccggcc gcggaggctg gatcggtccc ggtgtcttct
     2281 atggaggtca aaacagcgtg gatggcgtct ccaggcgatc tgacggttca ctaaacgagc
     2341 tctgcttata tagacctccc accgtacacg cctaccgccc atttgcgtca atggggcgga
     2401 gttgttacga cattttggaa agtcccgttg attttggtgc caaaacaaac tcccattgac
     2461 gtcaatgggg tggagacttg gaaatccccg tgagtcaaac cgctatccac gcccattgat
     2521 gtactgccaa aaccgcatca ccatggtaat agcgatgact aatacgtaga tgtactgcca
     2581 agtaggaaag tcccataagg tcatgtactg ggcataatgc caggcgggcc atttaccgtc
     2641 attgacgtca atagggggcg tacttggcat atgatacact tgatgtactg ccaagtgggc
     2701 agtttaccgt aaatactcca cccattgacg tcaatggaaa gtccctattg gcgttactat
     2761 gggaacatac gtcattattg acgtcaatgg gcgggggtcg ttgggcggtc agccaggcgg
     2821 gccatttacc gtaagttatg taacgcggaa ctccatatat gggctatgaa ctaatgaccc
     2881 cgtaattgat tactattaat aactagtcaa taatcaatgt cgggcgagct cgaattaatt
     2941 cgtaatcatg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     3001 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     3061 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     3121 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     3181 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     3241 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     3301 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     3361 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     3421 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     3481 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     3541 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     3601 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     3661 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     3721 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     3781 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     3841 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     3901 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     3961 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     4021 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     4081 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     4141 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     4201 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     4261 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     4321 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     4381 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     4441 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     4501 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     4561 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     4621 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     4681 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     4741 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     4801 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     4861 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     4921 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     4981 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     5041 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     5101 tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag
     5161 gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc
     5221 ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc
     5281 gtcagcgggt gttggcgggt gtcggggctg gcttaactat gcggcatcag agcagattgt
     5341 actgagagtg caccataaaa ttgtaaacgt taatattttg ttaaaattcg cgttaaattt
     5401 ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc
     5461 aaaagaatag cccgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt
     5521 aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact
     5581 acgtgaacca tcacccaaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg
     5641 gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag
     5701 aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac
     5761 gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtactatgg
     5821 ttgctttgac gtatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc
     5881 aggcgccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct
     5941 tcgctattac gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg
     6001 ccagggtttt cccagtcacg acgttgtaaa acgacggcca gtgccaagct gttaacagct
     6061 ttttgcaaaa gcctaggcct ccaaaaaagc ctcctcacta cttctggaat agctcagagg
     6121 ccgaggcg
//
