LOCUS       sequence-200428-        7832 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 7832)
  AUTHORS   Mercer CA, Kaliappan A, Dennis PB
  TITLE     Macroautophagy-dependent, intralysosomal cleavage of a betaine 
            homocysteine methyltransferase fusion protein requires stable 
            multimerization.
  JOURNAL   Autophagy. 2008 Feb;4(2):185-94. Epub 2007 Nov 7.
  PUBMED    18059170
REFERENCE   2  (bases 1 to 7832)
  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: 
            "Autophagy."; date: "2008-02"; volume: "4(2)"; pages: "185-94. Epub 
            2007 Nov 7"
COMMENT     Sequence Label: GST-BHMT-I/G
FEATURES             Location/Qualifiers
     source          1..7832
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        33..224
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     181..201
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     polyA_signal    293..427
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(317..336)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     371..390
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     regulatory      431..440
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(451..480)
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             complement(join(490..1200,1201..1203,1204..1206))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(490..1200,1201..1203,1204..1206))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(532..553)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     881..900
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1140..1161
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     misc_feature    1209..1758
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1368..1387)
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     primer_bind     1597..1614
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     complement(3013..3035)
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     primer_bind     complement(3694..3713)
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        complement(3726..3744)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3727..3744)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3954..3978)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        complement(3975..4178)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(4004..4024)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        4179..4558
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     complement(4587..4603)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4587..4603)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4600..4622)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4611..4627
                     /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(4635..4641,4642..4659,4660..4665))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4680..4701
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4818..4835)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4989..5577)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5069..5088)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5748..6539,6540..6608))
                     /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     6371..6390
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6609..6713)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6781..6799
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6837..6859)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6959..6978
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(6995..7450)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7132..7153)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7344..7363
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7648..7670
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7662..7679
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7663..7679
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        complement(join(7699..7832,1..224))
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      join(7713..7832,1..16)
                     /label=SV40 ori
                     /note="SV40 origin of replication"
ORIGIN
        1 gagttagggg cgggatgggc ggagttaggg gcgggactat ggttgctgac taattgagat
       61 gcatgctttg catacttctg cctgctgggg agcctgggga ctttccacac ctggttgctg
      121 actaattgag atgcatgctt tgcatacttc tgcctgctgg ggagcctggg gactttccac
      181 accctaactg acacacattc cacagctggt tctttccgcc tcagaaggta cctaaccaag
      241 ttcctctttc agaggttatt tcaggccatg gtgctgcgcc gaattaattc ccgatccaga
      301 catgataaga tacattgatg agtttggaca aaccacaact agaatgcagt gaaaaaaatg
      361 ctttatttgt gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa
      421 acaagttggg ccatggcggc caagctttca caggtcctcc tcggagatca gcttctgctc
      481 cattctagac ttgtacagct cgtccatgcc gagagtgatc ccggcggcgg tcacgaactc
      541 cagcaggacc atgtgatcgc gcttctcgtt ggggtctttg ctcagggcgg actgggtgct
      601 caggtagtgg ttgtcgggca gcagcacggg gccgtcgccg atgggggtgt tctgctggta
      661 gtggtcggcg agctgcacgc tgccgtcctc gatgttgtgg cggatcttga agttcacctt
      721 gatgccgttc ttctgcttgt cggccatgat atagacgttg tggctgttgt agttgtactc
      781 cagcttgtgc cccaggatgt tgccgtcctc cttgaagtcg atgcccttca gctcgatgcg
      841 gttcaccagg gtgtcgccct cgaacttcac ctcggcgcgg gtcttgtagt tgccgtcgtc
      901 cttgaagaag atggtgcgct cctggacgta gccttcgggc atggcggact tgaagaagtc
      961 gtgctgcttc atgtggtcgg ggtagcggct gaagcactgc acgccgtagg tcagggtggt
     1021 cacgagggtg ggccagggca cgggcagctt gccggtggtg cagatgaact tcagggtcag
     1081 cttgccgtag gtggcatcgc cctcgccctc gccggacacg ctgaacttgt ggccgtttac
     1141 gtcgccgtcc agctcgacca ggatgggcac caccccggtg aacagctcct cgcccttgct
     1201 caccatggta ttatcgtgtt tttcaaagga aaaccacgtc cccgtggttc ggggggccta
     1261 gacgtttttt taacctcgac taaacacatg taaagcatgt gcaccgaggc cccagatcag
     1321 atcccataca atggggtacc ttctgggcat ccttcagccc cttgttgaat acgcttgagg
     1381 agagccattt gactctttcc acaactatcc aactcacaac gtggcactgg ggttgtgccg
     1441 cctttgcagg tgtatcttat acacgtggct tttggccgca gaggcacctg tcgccaggtg
     1501 gggggttccg ctgcctgcaa agggtcgcta cagacgttgt ttgtcttcaa gaagcttcca
     1561 gaggaactgc ttccttcacg acattcaaca gaccttgcat tcctttggcg agaggggaaa
     1621 gacccctagg aatgctcgtc aagaagacag ggccaggttt ccgggccctc acattgccaa
     1681 aagacggcaa tatggtggaa aataacatat agacaaacgc acaccggcct tattccaagc
     1741 ggcttcggcc agtaacgtta gggggggggc ggaattgatc ccgctcgagg ttaacgaatt
     1801 cttatctaga gagctctttc agctgctgct cagttgtggc ttctttctgc tgcatcagct
     1861 cggctgttcc tttggtcact ccccagccat ctggctttga cattgaaggg ttgtatggcc
     1921 ggcctgaggc tatccgaaga ttctcccagt attccttcct ggcccttgct ctaacccagg
     1981 gtttggtgtg catgtccaaa ccacttcccc agctgccatg tttttctgaa gctggtggca
     2041 aaaagcccct ttctggggcc agctcctctg caattgccct gatgtggtag ggctcaaatc
     2101 cacagcaccc gccaatgtac ctgaccccca ggttgtaggc ctctctggcg tatttttgaa
     2161 tatcccatct ggtggcaact ctgggttcca gtccaaatgg gaattctggg agatcgatga
     2221 atccctgctt gttgcagtca ggagtgtggt aagccaaggg ctggctcatc aggtgagctt
     2281 tcagtcgggc agcctccaag ccctccttca tgagcttcac tgtttttaaa ctaatggtgg
     2341 ggtcaaagtg gcagttcaca ccaatgatgg atgctcctgc tttcaccagg cgcactgcac
     2401 actcgccggg gggcacgcca tgcaaatctc cttctgggcc aatgcacatg gttgctgcca
     2461 caggtttacc ggatgctatc aaggtttcaa ctgcccacac agcttcttca acgtgttcaa
     2521 aatactctgc aatcaagaag tccacgttct tcttcataaa gacctctaac tgttgcagaa
     2581 atactttttt gacttcagtt tcactcttgc agctaaggta tgaaggtgtc tgactcactc
     2641 ctcctgctac caaagcatct ccttcatcag ccacttgtcg ggcgatgtcg caagcagctt
     2701 cattgacttc ctgcccagat atcttctcta agacatagtt gcccctgttc tccagcttgt
     2761 cttcactcgc atagaaggtg aaggtctgca tgacgtttga gccagctctg aggaactctc
     2821 gatgaagctg gcgaactgct tctgggtgct ccacagcagc ttcaggagtc cagggtcctg
     2881 cctttacgta gcccctcttc tccagtgcaa agacaaaccc tccatctcca atcacaatct
     2941 ctccagcatt taaacgttct aggatgccct tcttggcgcg gccgccaccg gattgaggtg
     3001 ggtggccgcc accaccaaac gtggcttgcc agccctgcaa aggccatgct atatacttgc
     3061 tggatttcaa gtacttatca atttgtggga tagcttcaat acgtttttta aaacaaacta
     3121 attttgggaa cgcatccagg cacattgggt ccatgtataa aacaacatca agagcgtcat
     3181 acaacatgaa gtcaggatgg gttacatgat caccatttaa atatgtttta tgacataaac
     3241 gatcttcgaa cattttcagc atttcaggta gcttgctaag aaaatcaact ttgagagttt
     3301 caaagtcttt actatatgca attctcgaaa caccgtatct aatatccaaa accgctcctt
     3361 caagcattga aatctctgca cgctcttttg gacaaccacc caacatgttg tgcttgtcag
     3421 ctatataacg tatgatggcc atagactgtg ttaatttaac atcaccatca atataataag
     3481 gaagattggg aaactccaaa cccaattcaa actttttggc tcgccattta tcaccttcat
     3541 cgcgctcata caaatgctct tcatattttt cttcaagata ttccaaaaga agtcgagtgg
     3601 gttgcacaag gcccttaatt ttccaataac ctagtatagg ggacatggct ggctcgcgat
     3661 agaaccgagg tgcagttgga cctgggagtg gacacctgtg gagagaaagg caaagtggat
     3721 gttattctat agtgtcacct aaatcgtatg tgtatgatac ataaggttat gtattaattg
     3781 tagccgcgtt ctaacgaagc caagggggtg ggcctataga ctctataggc ggtacttacg
     3841 tcactcttgg cacggggaat ccgcgttcca atgcaccgtt cccggccgcg gaggctggat
     3901 cggtcccggt gtcttctatg gaggtcaaaa cagcgtggat ggcgtctcca ggcgatctga
     3961 cggttcacta aacgagctct gcttatatag acctcccacc gtacacgcct accgcccatt
     4021 tgcgtcaatg gggcggagtt gttacgacat tttggaaagt cccgttgatt ttggtgccaa
     4081 aacaaactcc cattgacgtc aatggggtgg agacttggaa atccccgtga gtcaaaccgc
     4141 tatccacgcc cattgatgta ctgccaaaac cgcatcacca tggtaatagc gatgactaat
     4201 acgtagatgt actgccaagt aggaaagtcc cataaggtca tgtactgggc ataatgccag
     4261 gcgggccatt taccgtcatt gacgtcaata gggggcgtac ttggcatatg atacacttga
     4321 tgtactgcca agtgggcagt ttaccgtaaa tactccaccc attgacgtca atggaaagtc
     4381 cctattggcg ttactatggg aacatacgtc attattgacg tcaatgggcg ggggtcgttg
     4441 ggcggtcagc caggcgggcc atttaccgta agttatgtaa cgcggaactc catatatggg
     4501 ctatgaacta atgaccccgt aattgattac tattaataac tagtcaataa tcaatgtcgg
     4561 gcgagctcga attaattcgt aatcatgtca tagctgtttc ctgtgtgaaa ttgttatccg
     4621 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     4681 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     4741 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     4801 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     4861 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     4921 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     4981 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     5041 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     5101 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     5161 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     5221 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     5281 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     5341 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     5401 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     5461 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     5521 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     5581 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     5641 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     5701 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     5761 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     5821 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     5881 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     5941 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     6001 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     6061 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     6121 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     6181 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     6241 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     6301 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     6361 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     6421 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     6481 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     6541 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     6601 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     6661 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     6721 ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa
     6781 aataggcgta tcacgaggcc ctttcgtctc gcgcgtttcg gtgatgacgg tgaaaacctc
     6841 tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga
     6901 caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggctggct taactatgcg
     6961 gcatcagagc agattgtact gagagtgcac cataaaattg taaacgttaa tattttgtta
     7021 aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc
     7081 aaaatccctt ataaatcaaa agaatagccc gagatagggt tgagtgttgt tccagtttgg
     7141 aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat
     7201 cagggcgatg gcccactacg tgaaccatca cccaaatcaa gttttttggg gtcgaggtgc
     7261 cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag
     7321 ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg
     7381 gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta
     7441 cagggcgcgt actatggttg ctttgacgta tgcggtgtga aataccgcac agatgcgtaa
     7501 ggagaaaata ccgcatcagg cgccattcgc cattcaggct gcgcaactgt tgggaagggc
     7561 gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc
     7621 gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg
     7681 ccaagctgtt aacagctttt tgcaaaagcc taggcctcca aaaaagcctc ctcactactt
     7741 ctggaatagc tcagaggccg aggcggcctc ggcctctgca taaataaaaa aaattagtca
     7801 gccatggggc ggagaatggg cggaactggg cg
//
