LOCUS       sequence-348296-        4910 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pTRE-EGFP-T2A-TFEB(delta30; amino acids 31-476)-polyA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4910)
  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 4910)
  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: 3927-64
FEATURES             Location/Qualifiers
     source          1..4910
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    55..73
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    91..109
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    127..145
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    163..181
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     226..250
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      325..334
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(331..333,334..336,337..1047)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(331..333,334..336,337..1047)
                     /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(376..397)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(637..656)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     984..1005
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1051..1104
                     /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    2552..3174
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2751..2770)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     rep_origin      complement(3323..3911)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3403..3422)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        4131..4233
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             4234..4893
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPELRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(4300..4319)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
ORIGIN
        1 taattgatta ctattaatac ctagttttcg tcttcaagaa ttcctcgagt ttactcccta
       61 tcagtgatag agaacgtatg accagtttac tccctatcag tgatagagaa cgtatctaca
      121 gtttactccc tatcagtgat agagaacgta tatccagttt actccctatc agtgatagag
      181 aacgtataag ctttaggcgt gtacggtggg cgcctataaa agcagagctc gtttagtgaa
      241 ccgtcagatc gcctggagca attccacaac acttttgtct tataccaact ttccgtacca
      301 cttcctaccc tcgtaaagcc tagggccacc atggtgagca agggcgagga gctgttcacc
      361 ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg
      421 tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc
      481 accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag
      541 tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc
      601 gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc
      661 gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac
      721 ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac
      781 gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac
      841 aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc
      901 gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa
      961 gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc
     1021 actctcggca tggacgagct gtacaagcgc gagggcagag gaagtcttct aacatgcggt
     1081 gacgtggagg agaatcccgg ccctatgcat tacatgcagc agcagcagca gcagcaacag
     1141 cagcagctcg gagggccgcc caccccggcc atcaataccc ccgtccactt ccagtcgcca
     1201 ccacctgtgc ctggggaggt gttgaaggtg cagtcctacc tggagaatcc cacatcctac
     1261 catctgcagc agtcgcagca tcagaaggtg cgggagtacc tgtccgagac ctatgggaac
     1321 aagtttgctg cccacatcag cccagcccag ggctctccga aacccccacc agccgcctcc
     1381 ccaggggtgc gagctggaca cgtgctgtcc tcctccgctg gcaacagtgc tcccaatagc
     1441 cccatggcca tgctgcacat tggctccaac cctgagaggg agttggatga tgtcattgac
     1501 aacattatgc gtctggacga tgtccttggc tacatcaatc ctgaaatgca gatgcccaac
     1561 acgctacccc tgtccagcag ccacctgaat gtgtacagca gcgaccccca ggtcacagcc
     1621 tccctggtgg gcgtcaccag cagctcctgc cctgcggacc tgacccagaa gcgagagctc
     1681 acagatgctg agagcagggc cctggccaag gagcggcaga agaaagacaa tcacaactta
     1741 attgaaagga gacgaaggtt caacatcaat gaccgcatca aggagttggg aatgctgatc
     1801 cccaaggcca atgacctgga cgtgcgctgg aacaagggca ccatcctcaa ggcctctgtg
     1861 gattacatcc ggaggatgca gaaggacctg caaaagtcca gggagctgga gaaccactct
     1921 cgccgcctgg agatgaccaa caagcagctc tggctccgta tccaggagct ggagatgcag
     1981 gctcgagtgc acggcctccc taccacctcc ccgtccggca tgaacatggc tgagctggcc
     2041 cagcaggtgg tgaagcagga gctgcctagc gaagagggcc caggggaggc cctgatgctg
     2101 ggggctgagg tccctgaccc tgagccactg ccagctctgc ccccgcaagc cccgctgccc
     2161 ctgcccaccc agccaccatc cccattccat cacctggact tcagccacag cctgagcttt
     2221 gggggcaggg aggacgaggg tcccccgggc taccccgaac ccctggcgcc ggggcatggc
     2281 tccccattcc ccagcctgtc caagaaggat ctggacctca tgctcctgga cgactcactg
     2341 ctaccgctgg cctctgatcc acttctgtcc accatgtccc ccgaggcctc caaggccagc
     2401 agccgccgga gcagcttcag catggaggag ggcgatgtgc tggaattcga ctacaaagac
     2461 catgacggcg attataaaga tcatgacatt gattacaagg atgacgatga ttaattaagt
     2521 aggtgagtaa ggatgtgcta cgaggtcgac cgggtggcat ccctgtgacc cctccccagt
     2581 gcctctcctg gccctggaag ttgccactcc agtgcccacc agccttgtcc taataaaatt
     2641 aagttgcatc attttgtctg actaggtgtc cttctataat attatggggt ggaggggggt
     2701 ggtatggagc aaggggcaag ttgggaagac aacctgtagg gcctgcgggg tctattggga
     2761 accaagctgg agtgcagtgg cacaatcttg gctcactgca atctccgcct cctgggttca
     2821 agcgattctc ctgcctcagc ctcccgagtt gttgggattc caggcatgca tgaccaggct
     2881 cagctaattt ttgttttttt ggtagagacg gggtttcacc atattggcca ggctggtctc
     2941 caactcctaa tctcaggtga tctacccacc ttggcctccc aaattgctgg gattacaggc
     3001 gtgaaccact gctcccttcc ctgtccttct gattttaaaa taactatacc agcaggagga
     3061 cgtccagaca cagcataggc tacctggcca tgcccaaccg gtgggacatt tgagttgctt
     3121 gcttggcact gtcctctcat gcgttgggtc cactcagtag atgcctgttg aattgggtac
     3181 gcggccagcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     3241 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     3301 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     3361 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     3421 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     3481 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     3541 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     3601 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     3661 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     3721 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     3781 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     3841 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     3901 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     3961 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     4021 tcgaccgaat aaatacctgt gacggaagat cacttcgcag aataaataaa tcctggtgtc
     4081 cctgttgata ccgggaagcc ctgggccaac ttttggcgaa aatgagacgt tgatcggcac
     4141 gtaagaggtt ccaactttca ccataatgaa ataagatcac taccgggcgt attttttgag
     4201 ttgtcgagat tttcaggagc taaggaagct aaaatggaga aaaaaatcac tggatatacc
     4261 accgttgata tatcccaatg gcatcgtaaa gaacattttg aggcatttca gtcagttgct
     4321 caatgtacct ataaccagac cgttcagctg gatattacgg cctttttaaa gaccgtaaag
     4381 aaaaataagc acaagtttta tccggccttt attcacattc ttgcccgcct gatgaatgct
     4441 catccggaat tacgtatggc aatgaaagac ggtgagctgg tgatatggga tagtgttcac
     4501 ccttgttaca ccgttttcca tgagcaaact gaaacgtttt catcgctctg gagtgaatac
     4561 cacgacgatt tccggcagtt tctacacata tattcgcaag atgtggcgtg ttacggtgaa
     4621 aacctggcct atttccctaa agggtttatt gagaatatgt ttttcgtctc agccaatccc
     4681 tgggtgagtt tcaccagttt tgatttaaac gtggccaata tggacaactt cttcgccccc
     4741 gttttcacca tgggcaaata ttatacgcaa ggcgacaagg tgctgatgcc gctggcgatt
     4801 caggttcatc atgccgtttg tgatggcttc catgtcggca gaatgcttaa tgaattacaa
     4861 cagtactgcg atgagtggca gggcggggcg taatacgcgt aatgaccccg
//
