LOCUS       sequence-348292-        5000 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pTRE-EGFP-T2A-TFEB(S3A)-polyA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5000)
  AUTHORS   Hsu CL, Lee EX, Gordon KL, Paz EA, Shen WC, Ohnishi K, Meisenhelder 
            J, Hunter T, La Spada AR
  TITLE     MAP4K3 mediates amino acid-dependent regulation of autophagy via 
            phosphorylation of TFEB.
  JOURNAL   Nat Commun. 2018 Mar 5;9(1):942. doi: 10.1038/s41467-018-03340-7.
  PUBMED    29507340
REFERENCE   2  (bases 1 to 5000)
  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: "Nat 
            Commun."; date: "2018-03-5"; volume: "9(1):942. doi"; pages: " 
            10.1038/s41467-018-03340-7"
COMMENT     Sequence Label: 3923-51
FEATURES             Location/Qualifiers
     source          1..5000
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1..660
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPELRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(67..86)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     protein_bind    732..750
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    768..786
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    804..822
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    840..858
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     903..927
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      1002..1011
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1008..1010,1011..1013,1014..1724)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1008..1010,1011..1013,1014..1724)
                     /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(1053..1074)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1314..1333)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1661..1682
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1728..1781
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     polyA_signal    3319..3941
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3518..3537)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     rep_origin      complement(4090..4678)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4170..4189)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        4898..5000
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
ORIGIN
        1 atggagaaaa aaatcactgg atataccacc gttgatatat cccaatggca tcgtaaagaa
       61 cattttgagg catttcagtc agttgctcaa tgtacctata accagaccgt tcagctggat
      121 attacggcct ttttaaagac cgtaaagaaa aataagcaca agttttatcc ggcctttatt
      181 cacattcttg cccgcctgat gaatgctcat ccggaattac gtatggcaat gaaagacggt
      241 gagctggtga tatgggatag tgttcaccct tgttacaccg ttttccatga gcaaactgaa
      301 acgttttcat cgctctggag tgaataccac gacgatttcc ggcagtttct acacatatat
      361 tcgcaagatg tggcgtgtta cggtgaaaac ctggcctatt tccctaaagg gtttattgag
      421 aatatgtttt tcgtctcagc caatccctgg gtgagtttca ccagttttga tttaaacgtg
      481 gccaatatgg acaacttctt cgcccccgtt ttcaccatgg gcaaatatta tacgcaaggc
      541 gacaaggtgc tgatgccgct ggcgattcag gttcatcatg ccgtttgtga tggcttccat
      601 gtcggcagaa tgcttaatga attacaacag tactgcgatg agtggcaggg cggggcgtaa
      661 tacgcgtaat gaccccgtaa ttgattacta ttaataccta gttttcgtct tcaagaattc
      721 ctcgagttta ctccctatca gtgatagaga acgtatgacc agtttactcc ctatcagtga
      781 tagagaacgt atctacagtt tactccctat cagtgataga gaacgtatat ccagtttact
      841 ccctatcagt gatagagaac gtataagctt taggcgtgta cggtgggcgc ctataaaagc
      901 agagctcgtt tagtgaaccg tcagatcgcc tggagcaatt ccacaacact tttgtcttat
      961 accaactttc cgtaccactt cctaccctcg taaagcctag ggccaccatg gtgagcaagg
     1021 gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg
     1081 gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc
     1141 tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc
     1201 tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct
     1261 tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg
     1321 gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg
     1381 agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca
     1441 actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga
     1501 acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc
     1561 agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc
     1621 agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg
     1681 tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagcgcgag ggcagaggaa
     1741 gtcttctaac atgcggtgac gtggaggaga atcccggccc tatggcggca cgcatagggt
     1801 tgcgcatgca gctcatgcgg gagcaggcgc agcaggagga gcagcgggag cgcatgcagc
     1861 aacaggctgt catgcattac atgcagcagc agcagcagca gcaacagcag cagctcggag
     1921 ggccgcccac cccggccatc aatacccccg tccacttcca gtcgccacca cctgtgcctg
     1981 gggaggtgtt gaaggtgcag tcctacctgg agaatcccac atcctaccat ctgcagcagt
     2041 cgcagcatca gaaggtgcgg gagtacctgt ccgagaccta tgggaacaag tttgctgccc
     2101 acatcagccc agcccagggc tctccgaaac ccccaccagc cgcctcccca ggggtgcgag
     2161 ctggacacgt gctgtcctcc tccgctggca acagtgctcc caatagcccc atggccatgc
     2221 tgcacattgg ctccaaccct gagagggagt tggatgatgt cattgacaac attatgcgtc
     2281 tggacgatgt ccttggctac atcaatcctg aaatgcagat gcccaacacg ctacccctgt
     2341 ccagcagcca cctgaatgtg tacagcagcg acccccaggt cacagcctcc ctggtgggcg
     2401 tcaccagcag ctcctgccct gcggacctga cccagaagcg agagctcaca gatgctgaga
     2461 gcagggccct ggccaaggag cggcagaaga aagacaatca caacttaatt gaaaggagac
     2521 gaaggttcaa catcaatgac cgcatcaagg agttgggaat gctgatcccc aaggccaatg
     2581 acctggacgt gcgctggaac aagggcacca tcctcaaggc ctctgtggat tacatccgga
     2641 ggatgcagaa ggacctgcaa aagtccaggg agctggagaa ccactctcgc cgcctggaga
     2701 tgaccaacaa gcagctctgg ctccgtatcc aggagctgga gatgcaggct cgagtgcacg
     2761 gcctccctac cacctccccg tccggcatga acatggctga gctggcccag caggtggtga
     2821 agcaggagct gcctagcgaa gagggcccag gggaggccct gatgctgggg gctgaggtcc
     2881 ctgaccctga gccactgcca gctctgcccc cgcaagcccc gctgcccctg cccacccagc
     2941 caccatcccc attccatcac ctggacttca gccacagcct gagctttggg ggcagggagg
     3001 acgagggtcc cccgggctac cccgaacccc tggcgccggg gcatggctcc ccattcccca
     3061 gcctgtccaa gaaggatctg gacctcatgc tcctggacga ctcactgcta ccgctggcct
     3121 ctgatccact tctgtccacc atgtcccccg aggcctccaa ggccagcagc cgccggagca
     3181 gcttcagcat ggaggagggc gatgtgctgg aattcgacta caaagaccat gacggcgatt
     3241 ataaagatca tgacattgat tacaaggatg acgatgatta attaagtagg tgagtaagga
     3301 tgtgctacga ggtcgaccgg gtggcatccc tgtgacccct ccccagtgcc tctcctggcc
     3361 ctggaagttg ccactccagt gcccaccagc cttgtcctaa taaaattaag ttgcatcatt
     3421 ttgtctgact aggtgtcctt ctataatatt atggggtgga ggggggtggt atggagcaag
     3481 gggcaagttg ggaagacaac ctgtagggcc tgcggggtct attgggaacc aagctggagt
     3541 gcagtggcac aatcttggct cactgcaatc tccgcctcct gggttcaagc gattctcctg
     3601 cctcagcctc ccgagttgtt gggattccag gcatgcatga ccaggctcag ctaatttttg
     3661 tttttttggt agagacgggg tttcaccata ttggccaggc tggtctccaa ctcctaatct
     3721 caggtgatct acccaccttg gcctcccaaa ttgctgggat tacaggcgtg aaccactgct
     3781 cccttccctg tccttctgat tttaaaataa ctataccagc aggaggacgt ccagacacag
     3841 cataggctac ctggccatgc ccaaccggtg ggacatttga gttgcttgct tggcactgtc
     3901 ctctcatgcg ttgggtccac tcagtagatg cctgttgaat tgggtacgcg gccagcggcg
     3961 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     4021 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     4081 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     4141 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     4201 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     4261 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     4321 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     4381 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     4441 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     4501 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     4561 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     4621 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     4681 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     4741 gattttggtc atgagattat caaaaaggat cttcacctag atccttttcg accgaataaa
     4801 tacctgtgac ggaagatcac ttcgcagaat aaataaatcc tggtgtccct gttgataccg
     4861 ggaagccctg ggccaacttt tggcgaaaat gagacgttga tcggcacgta agaggttcca
     4921 actttcacca taatgaaata agatcactac cgggcgtatt ttttgagttg tcgagatttt
     4981 caggagctaa ggaagctaaa
//
