LOCUS       sequence-348293-        5000 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pTRE-EGFP-T2A-TFEB(wt)-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: 3922-48
FEATURES             Location/Qualifiers
     source          1..5000
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     167..188
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             234..287
                     /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    1825..2447
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2024..2043)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     rep_origin      complement(2596..3184)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2676..2695)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        3404..3506
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             3507..4166
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPELRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(3573..3592)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     protein_bind    4238..4256
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4274..4292
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4310..4328
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4346..4364
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     4409..4433
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      4508..4517
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(4514..4516,4517..4519,4520..5000,1..230)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(4514..4516,4517..4519,4520..5000,1..230)
                     /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(4559..4580)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4820..4839)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
ORIGIN
        1 aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact
       61 accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga
      121 gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg
      181 agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag cgcgagggca
      241 gaggaagtct tctaacatgc ggtgacgtgg aggagaatcc cggccctatg gcgtcacgca
      301 tagggttgcg catgcagctc atgcgggagc aggcgcagca ggaggagcag cgggagcgca
      361 tgcagcaaca ggctgtcatg cattacatgc agcagcagca gcagcagcaa cagcagcagc
      421 tcggagggcc gcccaccccg gccatcaata cccccgtcca cttccagtcg ccaccacctg
      481 tgcctgggga ggtgttgaag gtgcagtcct acctggagaa tcccacatcc taccatctgc
      541 agcagtcgca gcatcagaag gtgcgggagt acctgtccga gacctatggg aacaagtttg
      601 ctgcccacat cagcccagcc cagggctctc cgaaaccccc accagccgcc tccccagggg
      661 tgcgagctgg acacgtgctg tcctcctccg ctggcaacag tgctcccaat agccccatgg
      721 ccatgctgca cattggctcc aaccctgaga gggagttgga tgatgtcatt gacaacatta
      781 tgcgtctgga cgatgtcctt ggctacatca atcctgaaat gcagatgccc aacacgctac
      841 ccctgtccag cagccacctg aatgtgtaca gcagcgaccc ccaggtcaca gcctccctgg
      901 tgggcgtcac cagcagctcc tgccctgcgg acctgaccca gaagcgagag ctcacagatg
      961 ctgagagcag ggccctggcc aaggagcggc agaagaaaga caatcacaac ttaattgaaa
     1021 ggagacgaag gttcaacatc aatgaccgca tcaaggagtt gggaatgctg atccccaagg
     1081 ccaatgacct ggacgtgcgc tggaacaagg gcaccatcct caaggcctct gtggattaca
     1141 tccggaggat gcagaaggac ctgcaaaagt ccagggagct ggagaaccac tctcgccgcc
     1201 tggagatgac caacaagcag ctctggctcc gtatccagga gctggagatg caggctcgag
     1261 tgcacggcct ccctaccacc tccccgtccg gcatgaacat ggctgagctg gcccagcagg
     1321 tggtgaagca ggagctgcct agcgaagagg gcccagggga ggccctgatg ctgggggctg
     1381 aggtccctga ccctgagcca ctgccagctc tgcccccgca agccccgctg cccctgccca
     1441 cccagccacc atccccattc catcacctgg acttcagcca cagcctgagc tttgggggca
     1501 gggaggacga gggtcccccg ggctaccccg aacccctggc gccggggcat ggctccccat
     1561 tccccagcct gtccaagaag gatctggacc tcatgctcct ggacgactca ctgctaccgc
     1621 tggcctctga tccacttctg tccaccatgt cccccgaggc ctccaaggcc agcagccgcc
     1681 ggagcagctt cagcatggag gagggcgatg tgctggaatt cgactacaaa gaccatgacg
     1741 gcgattataa agatcatgac attgattaca aggatgacga tgattaatta agtaggtgag
     1801 taaggatgtg ctacgaggtc gaccgggtgg catccctgtg acccctcccc agtgcctctc
     1861 ctggccctgg aagttgccac tccagtgccc accagccttg tcctaataaa attaagttgc
     1921 atcattttgt ctgactaggt gtccttctat aatattatgg ggtggagggg ggtggtatgg
     1981 agcaaggggc aagttgggaa gacaacctgt agggcctgcg gggtctattg ggaaccaagc
     2041 tggagtgcag tggcacaatc ttggctcact gcaatctccg cctcctgggt tcaagcgatt
     2101 ctcctgcctc agcctcccga gttgttggga ttccaggcat gcatgaccag gctcagctaa
     2161 tttttgtttt tttggtagag acggggtttc accatattgg ccaggctggt ctccaactcc
     2221 taatctcagg tgatctaccc accttggcct cccaaattgc tgggattaca ggcgtgaacc
     2281 actgctccct tccctgtcct tctgatttta aaataactat accagcagga ggacgtccag
     2341 acacagcata ggctacctgg ccatgcccaa ccggtgggac atttgagttg cttgcttggc
     2401 actgtcctct catgcgttgg gtccactcag tagatgcctg ttgaattggg tacgcggcca
     2461 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     2521 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     2581 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     2641 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     2701 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     2761 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     2821 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     2881 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     2941 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     3001 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     3061 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     3121 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     3181 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     3241 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttcgaccg
     3301 aataaatacc tgtgacggaa gatcacttcg cagaataaat aaatcctggt gtccctgttg
     3361 ataccgggaa gccctgggcc aacttttggc gaaaatgaga cgttgatcgg cacgtaagag
     3421 gttccaactt tcaccataat gaaataagat cactaccggg cgtatttttt gagttgtcga
     3481 gattttcagg agctaaggaa gctaaaatgg agaaaaaaat cactggatat accaccgttg
     3541 atatatccca atggcatcgt aaagaacatt ttgaggcatt tcagtcagtt gctcaatgta
     3601 cctataacca gaccgttcag ctggatatta cggccttttt aaagaccgta aagaaaaata
     3661 agcacaagtt ttatccggcc tttattcaca ttcttgcccg cctgatgaat gctcatccgg
     3721 aattacgtat ggcaatgaaa gacggtgagc tggtgatatg ggatagtgtt cacccttgtt
     3781 acaccgtttt ccatgagcaa actgaaacgt tttcatcgct ctggagtgaa taccacgacg
     3841 atttccggca gtttctacac atatattcgc aagatgtggc gtgttacggt gaaaacctgg
     3901 cctatttccc taaagggttt attgagaata tgtttttcgt ctcagccaat ccctgggtga
     3961 gtttcaccag ttttgattta aacgtggcca atatggacaa cttcttcgcc cccgttttca
     4021 ccatgggcaa atattatacg caaggcgaca aggtgctgat gccgctggcg attcaggttc
     4081 atcatgccgt ttgtgatggc ttccatgtcg gcagaatgct taatgaatta caacagtact
     4141 gcgatgagtg gcagggcggg gcgtaatacg cgtaatgacc ccgtaattga ttactattaa
     4201 tacctagttt tcgtcttcaa gaattcctcg agtttactcc ctatcagtga tagagaacgt
     4261 atgaccagtt tactccctat cagtgataga gaacgtatct acagtttact ccctatcagt
     4321 gatagagaac gtatatccag tttactccct atcagtgata gagaacgtat aagctttagg
     4381 cgtgtacggt gggcgcctat aaaagcagag ctcgtttagt gaaccgtcag atcgcctgga
     4441 gcaattccac aacacttttg tcttatacca actttccgta ccacttccta ccctcgtaaa
     4501 gcctagggcc accatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct
     4561 ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg
     4621 cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt
     4681 gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc
     4741 cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga
     4801 gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga
     4861 gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa
     4921 catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga
     4981 caagcagaag aacggcatca
//
