LOCUS       sequence-348295-        5000 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pTRE-EGFP-T2A-TFEB(S3E)-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: 3924-55
FEATURES             Location/Qualifiers
     source          1..5000
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    65..83
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    101..119
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    137..155
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    173..191
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     236..260
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      335..344
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(341..343,344..346,347..1057)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(341..343,344..346,347..1057)
                     /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(386..407)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(647..666)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     994..1015
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1061..1114
                     /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    2652..3274
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2851..2870)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     rep_origin      complement(3423..4011)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3503..3522)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        4231..4333
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             4334..4993
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPELRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(4400..4419)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
ORIGIN
        1 aatgaccccg taattgatta ctattaatac ctagttttcg tcttcaagaa ttcctcgagt
       61 ttactcccta tcagtgatag agaacgtatg accagtttac tccctatcag tgatagagaa
      121 cgtatctaca gtttactccc tatcagtgat agagaacgta tatccagttt actccctatc
      181 agtgatagag aacgtataag ctttaggcgt gtacggtggg cgcctataaa agcagagctc
      241 gtttagtgaa ccgtcagatc gcctggagca attccacaac acttttgtct tataccaact
      301 ttccgtacca cttcctaccc tcgtaaagcc tagggccacc atggtgagca agggcgagga
      361 gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa
      421 gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt
      481 catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta
      541 cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc
      601 cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta
      661 caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa
      721 gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa
      781 cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa
      841 gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac
      901 ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc
      961 cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc
     1021 cgccgggatc actctcggca tggacgagct gtacaagcgc gagggcagag gaagtcttct
     1081 aacatgcggt gacgtggagg agaatcccgg ccctatggcg gaacgcatag ggttgcgcat
     1141 gcagctcatg cgggagcagg cgcagcagga ggagcagcgg gagcgcatgc agcaacaggc
     1201 tgtcatgcat tacatgcagc agcagcagca gcagcaacag cagcagctcg gagggccgcc
     1261 caccccggcc atcaataccc ccgtccactt ccagtcgcca ccacctgtgc ctggggaggt
     1321 gttgaaggtg cagtcctacc tggagaatcc cacatcctac catctgcagc agtcgcagca
     1381 tcagaaggtg cgggagtacc tgtccgagac ctatgggaac aagtttgctg cccacatcag
     1441 cccagcccag ggctctccga aacccccacc agccgcctcc ccaggggtgc gagctggaca
     1501 cgtgctgtcc tcctccgctg gcaacagtgc tcccaatagc cccatggcca tgctgcacat
     1561 tggctccaac cctgagaggg agttggatga tgtcattgac aacattatgc gtctggacga
     1621 tgtccttggc tacatcaatc ctgaaatgca gatgcccaac acgctacccc tgtccagcag
     1681 ccacctgaat gtgtacagca gcgaccccca ggtcacagcc tccctggtgg gcgtcaccag
     1741 cagctcctgc cctgcggacc tgacccagaa gcgagagctc acagatgctg agagcagggc
     1801 cctggccaag gagcggcaga agaaagacaa tcacaactta attgaaagga gacgaaggtt
     1861 caacatcaat gaccgcatca aggagttggg aatgctgatc cccaaggcca atgacctgga
     1921 cgtgcgctgg aacaagggca ccatcctcaa ggcctctgtg gattacatcc ggaggatgca
     1981 gaaggacctg caaaagtcca gggagctgga gaaccactct cgccgcctgg agatgaccaa
     2041 caagcagctc tggctccgta tccaggagct ggagatgcag gctcgagtgc acggcctccc
     2101 taccacctcc ccgtccggca tgaacatggc tgagctggcc cagcaggtgg tgaagcagga
     2161 gctgcctagc gaagagggcc caggggaggc cctgatgctg ggggctgagg tccctgaccc
     2221 tgagccactg ccagctctgc ccccgcaagc cccgctgccc ctgcccaccc agccaccatc
     2281 cccattccat cacctggact tcagccacag cctgagcttt gggggcaggg aggacgaggg
     2341 tcccccgggc taccccgaac ccctggcgcc ggggcatggc tccccattcc ccagcctgtc
     2401 caagaaggat ctggacctca tgctcctgga cgactcactg ctaccgctgg cctctgatcc
     2461 acttctgtcc accatgtccc ccgaggcctc caaggccagc agccgccgga gcagcttcag
     2521 catggaggag ggcgatgtgc tggaattcga ctacaaagac catgacggcg attataaaga
     2581 tcatgacatt gattacaagg atgacgatga ttaattaagt aggtgagtaa ggatgtgcta
     2641 cgaggtcgac cgggtggcat ccctgtgacc cctccccagt gcctctcctg gccctggaag
     2701 ttgccactcc agtgcccacc agccttgtcc taataaaatt aagttgcatc attttgtctg
     2761 actaggtgtc cttctataat attatggggt ggaggggggt ggtatggagc aaggggcaag
     2821 ttgggaagac aacctgtagg gcctgcgggg tctattggga accaagctgg agtgcagtgg
     2881 cacaatcttg gctcactgca atctccgcct cctgggttca agcgattctc ctgcctcagc
     2941 ctcccgagtt gttgggattc caggcatgca tgaccaggct cagctaattt ttgttttttt
     3001 ggtagagacg gggtttcacc atattggcca ggctggtctc caactcctaa tctcaggtga
     3061 tctacccacc ttggcctccc aaattgctgg gattacaggc gtgaaccact gctcccttcc
     3121 ctgtccttct gattttaaaa taactatacc agcaggagga cgtccagaca cagcataggc
     3181 tacctggcca tgcccaaccg gtgggacatt tgagttgctt gcttggcact gtcctctcat
     3241 gcgttgggtc cactcagtag atgcctgttg aattgggtac gcggccagcg gcgagcggta
     3301 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     3361 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     3421 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     3481 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     3541 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     3601 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     3661 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     3721 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     3781 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     3841 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     3901 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     3961 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     4021 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     4081 gtcatgagat tatcaaaaag gatcttcacc tagatccttt tcgaccgaat aaatacctgt
     4141 gacggaagat cacttcgcag aataaataaa tcctggtgtc cctgttgata ccgggaagcc
     4201 ctgggccaac ttttggcgaa aatgagacgt tgatcggcac gtaagaggtt ccaactttca
     4261 ccataatgaa ataagatcac taccgggcgt attttttgag ttgtcgagat tttcaggagc
     4321 taaggaagct aaaatggaga aaaaaatcac tggatatacc accgttgata tatcccaatg
     4381 gcatcgtaaa gaacattttg aggcatttca gtcagttgct caatgtacct ataaccagac
     4441 cgttcagctg gatattacgg cctttttaaa gaccgtaaag aaaaataagc acaagtttta
     4501 tccggccttt attcacattc ttgcccgcct gatgaatgct catccggaat tacgtatggc
     4561 aatgaaagac ggtgagctgg tgatatggga tagtgttcac ccttgttaca ccgttttcca
     4621 tgagcaaact gaaacgtttt catcgctctg gagtgaatac cacgacgatt tccggcagtt
     4681 tctacacata tattcgcaag atgtggcgtg ttacggtgaa aacctggcct atttccctaa
     4741 agggtttatt gagaatatgt ttttcgtctc agccaatccc tgggtgagtt tcaccagttt
     4801 tgatttaaac gtggccaata tggacaactt cttcgccccc gttttcacca tgggcaaata
     4861 ttatacgcaa ggcgacaagg tgctgatgcc gctggcgatt caggttcatc atgccgtttg
     4921 tgatggcttc catgtcggca gaatgcttaa tgaattacaa cagtactgcg atgagtggca
     4981 gggcggggcg taatacgcgt
//
