LOCUS       sequence-197985-        6122 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 6122)
  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 6122)
  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..6122
                     /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      2422..2431
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2435..2569
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2472..2491)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2526..2545
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        2638..2995
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2638..2829
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2661..2681)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2846..2981
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3015..3032)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3015..3031)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3024..3046)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3244..3699
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3331..3350)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3541..3562
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3716..3735)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3835..3857
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3895..3913)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3981..4085
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4086..4154,4155..4946)
                     /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(4304..4323)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5117..5705
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5606..5625
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5859..5876
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5993..6014
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6029..6034,6035..6052,6053..6059)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6067..6083
                     /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     6072..6094
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6091..6107
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6091..6107
                     /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 tctagaatca ctactgtcga gtctaattcc agttggtgga
     2341 ccaactgggt gatcccagcc atctcagccc tggtggtagc tctgatgtat cgcctctaca
     2401 tggcagaaga ttagaagctt ggccgccatg gcccaacttg tttattgcag cttataatgg
     2461 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
     2521 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggatcg ggaattaatt
     2581 cggcgcagca ccatggcctg aaataacctc tgaaagagga acttggttag gtaccttctg
     2641 aggcggaaag aaccagctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc
     2701 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa
     2761 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     2821 catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc
     2881 tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc
     2941 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct
     3001 gttaacagct tggcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt
     3061 acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag
     3121 gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg gcgcctgatg
     3181 cggtattttc tccttacgca tctgtgcggt atttcacacc gcatacgtca aagcaaccat
     3241 agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     3301 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     3361 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     3421 ttagtgcttt acggcacctc gaccccaaaa aacttgattt gggtgatggt tcacgtagtg
     3481 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     3541 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcgggctat tcttttgatt
     3601 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     3661 ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt atggtgcact ctcagtacaa
     3721 tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc
     3781 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
     3841 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg
     3901 tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg
     3961 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     4021 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     4081 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     4141 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     4201 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     4261 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     4321 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     4381 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     4441 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     4501 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     4561 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     4621 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     4681 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     4741 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     4801 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     4861 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     4921 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     4981 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     5041 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     5101 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     5161 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     5221 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
     5281 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     5341 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     5401 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     5461 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     5521 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     5581 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     5641 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     5701 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     5761 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     5821 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     5881 cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca
     5941 gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga
     6001 gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt
     6061 gtggaattgt gagcggataa caatttcaca caggaaacag ctatgacatg attacgaatt
     6121 aa
//
