LOCUS       sequence-348294-        4197 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pTRE-EGFP-T2A-mCherry-polyA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4197)
  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 4197)
  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: 3918-37
FEATURES             Location/Qualifiers
     source          1..4197
                     /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"
     CDS             1791..2498
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(1933..1951)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2201..2220
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     RBS             2502..2510
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     polyA_signal    2516..3138
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2715..2734)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     rep_origin      complement(3287..3875)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3367..3386)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        4095..4197
                     /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 tatgctagcc gtgagcaagg
     1801 gcgaggagga taacatggcc atcatcaagg agttcatgcg cttcaaggtg cacatggagg
     1861 gctccgtgaa cggccacgag ttcgagatcg agggcgaggg cgagggccgc ccctacgagg
     1921 gcacccagac cgccaagctg aaggtgacca agggtggccc cctgcccttc gcctgggaca
     1981 tcctgtcccc tcagttcatg tacggctcca aggcctacgt gaagcacccc gccgacatcc
     2041 ccgactactt gaagctgtcc ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg
     2101 aggacggcgg cgtggtgacc gtgacccagg actcctccct gcaggacggc gagttcatct
     2161 acaaggtgaa gctgcgcggc accaacttcc cctccgacgg ccccgtaatg cagaagaaga
     2221 ccatgggctg ggaggcctcc tccgagcgga tgtaccccga ggacggcgcc ctgaagggcg
     2281 agatcaagca gaggctgaag ctgaaggacg gcggccacta cgacgctgag gtcaagacca
     2341 cctacaaggc caagaagccc gtgcagctgc ccggcgccta caacgtcaac atcaagttgg
     2401 acatcacctc ccacaacgag gactacacca tcgtggaaca gtacgaacgc gccgagggcc
     2461 gccactccac cggcggcatg gacgagctgt acaagtagtc ttaaggaggt cgaccgggtg
     2521 gcatccctgt gacccctccc cagtgcctct cctggccctg gaagttgcca ctccagtgcc
     2581 caccagcctt gtcctaataa aattaagttg catcattttg tctgactagg tgtccttcta
     2641 taatattatg gggtggaggg gggtggtatg gagcaagggg caagttggga agacaacctg
     2701 tagggcctgc ggggtctatt gggaaccaag ctggagtgca gtggcacaat cttggctcac
     2761 tgcaatctcc gcctcctggg ttcaagcgat tctcctgcct cagcctcccg agttgttggg
     2821 attccaggca tgcatgacca ggctcagcta atttttgttt ttttggtaga gacggggttt
     2881 caccatattg gccaggctgg tctccaactc ctaatctcag gtgatctacc caccttggcc
     2941 tcccaaattg ctgggattac aggcgtgaac cactgctccc ttccctgtcc ttctgatttt
     3001 aaaataacta taccagcagg aggacgtcca gacacagcat aggctacctg gccatgccca
     3061 accggtggga catttgagtt gcttgcttgg cactgtcctc tcatgcgttg ggtccactca
     3121 gtagatgcct gttgaattgg gtacgcggcc agcggcgagc ggtatcagct cactcaaagg
     3181 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     3241 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     3301 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     3361 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     3421 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     3481 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     3541 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     3601 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     3661 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     3721 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     3781 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     3841 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     3901 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     3961 aaaggatctt cacctagatc cttttcgacc gaataaatac ctgtgacgga agatcacttc
     4021 gcagaataaa taaatcctgg tgtccctgtt gataccggga agccctgggc caacttttgg
     4081 cgaaaatgag acgttgatcg gcacgtaaga ggttccaact ttcaccataa tgaaataaga
     4141 tcactaccgg gcgtattttt tgagttgtcg agattttcag gagctaagga agctaaa
//
