LOCUS       sequence-320294-        7852 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For mammalian expression of a Flag-tagged, constitutively active 
            version (S142A, S211A) of Tfeb..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7852)
  AUTHORS   Young NP, Kamireddy A, Van Nostrand JL, Eichner LJ, Shokhirev MN, 
            Dayn Y, Shaw RJ
  TITLE     AMPK governs lineage specification through Tfeb-dependent regulation
            of lysosomes.
  JOURNAL   Genes Dev. 2016 Mar 1;30(5):535-52. doi: 10.1101/gad.274142.115.
  PUBMED    26944679
REFERENCE   2  (bases 1 to 7852)
  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"; doi: 
            "10.1101/gad.274142"; journalName: "Genes Dev"; date: "2016-03-1- 
            1"; volume: "30"; issue: "5"; pages: "535-52"
COMMENT     Sequence Label: pCIP-Flag-TfebAA
FEATURES             Location/Qualifiers
     source          1..7852
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          658..1674
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1598..1620
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1682..1701
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1741..1750
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1750..1773
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     misc_feature    join(3453..4027,4028..4030,4031..4039)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3620..3637)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3847..3866
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4040..4639
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(4040..4059)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4536..4556
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(4720..4737)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4726..4933
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4987..5007)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4987..5005)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5026..5042)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5026..5042)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5039..5061)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5050..5066
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(5074..5080,5081..5098,5099..5104))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5119..5140
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5257..5274)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5428..6016)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5508..5527)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6187..6978,6979..7047))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6810..6829
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7048..7152)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      7179..7635
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7266..7285)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7476..7497
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7761..7783
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7775..7792
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7776..7792
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7802..7821
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        7802..7820
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtcgaggtga gccccacgtt ctgcttcact ctccccatct
      421 cccccccctc cccaccccca attttgtatt tatttatttt ttaattattt tgtgcagcga
      481 tgggggcggg gggggggggg gsgcgcgccr gggggggggg gggggggggg gggggggggg
      541 ggggsgrggc ggagaggtgc ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct
      601 tttatggcga ggcggcggcg gcggcggccc tataaaaagc gaagcgcgcg gcgggcggga
      661 gtcgctgcgc gctgccttcg ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc
      721 cggctctgac tgaccgcgtt actcccacag gtgagcgggc gggacggccc ttctcctccg
      781 ggctgtaatt agcgcttggt ttaatgacgg cttgtttctt ttctgtggct gcgtgaaagc
      841 cttgaggggc tccgggaggg ccctttgtgc ggggggagcg gctcgggggg tgcgtgcgtg
      901 tgtgtgtgcg tggggagcgc cgcgtgcggc tccgcgctgc ccggcggctg tgagcgctgc
      961 gggcgcggcg cggggctttg tgcgctccgc agtgtgcgcg aggggagcgc ggccgggggc
     1021 ggtgccccgc ggtgcggggg gggctgcgag gggaacaaag gctgcgtgcg gggtgtgtgc
     1081 gtgggggggt gagcaggggg tgtgggcgcg tcggtcgggc tgcaaccccc cctgcacccc
     1141 cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtac ggggcgtggc
     1201 gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg
     1261 ccgcctcggg ccggggrggg ctcgggggag gggcgcggcg gcccccggag cgccggcggc
     1321 tgtcgaggcg cggcgagccg cagccattgc cttttatggt aatcgtgcga gagggcgcag
     1381 ggacttcctt tgtcccaaat ctgtgcggag ccgaaatctg ggaggcgccg ccgcaccccc
     1441 tctagcgggc gcggggcgaa gcggtgcggc gccggcagga aggaaatggg cggggagggc
     1501 cttcgtgcgt cgccgcgccg ccgtcccctt ctccctctcc agcctcgggg ctgtccgcgg
     1561 ggggacggct gccttcgggg gggacggggc agggcggggt tcggcttctg gcgtgtgacc
     1621 ggcggctcta gagcctctgc taaccatgtt catgccttct tctttttcct acagctcctg
     1681 ggcaacgtgc tggttattgt gctgtctcat cattttggca aagaattcaa cgcggctagc
     1741 gccaccatgg attacaagga cgatgacgac aaactcggat ccatgggaac caattcagtc
     1801 gacgctcagc tcgctcagtg gtcttgggca aatcccttct gcccggactc agtttctcct
     1861 tgtgcacaat gggagcaacc atacttatgc cagcctgtgc ttaaagacta cgaagatgat
     1921 gaatacttca tgggcctgtc tcccctcgac tacagggagc ccgaaccaac agctgccatg
     1981 gcgtcacgca tcgggctgcg catgcagctc atgcgggagc aggcccagca ggaggagcag
     2041 cgagagcgca tgcagcagca ggctgtcatg cattatatgc aacagcagca gcagcagcag
     2101 cagcagctgg gtgggccccc caccccagcc atcaacaccc ctgtccactt ccagtcgccc
     2161 ccgcctgtgc ccggggaggt gctgaaggtg cagtcctacc tggagaaccc cacctcctac
     2221 cacctgcaac agtcccagca tcagaaggtt cgggagtatc tgtctgagac ctatgggaac
     2281 aagtttgctg cccacgtgag cccagcccaa ggttccccga agcctgcccc agcagcatcc
     2341 ccaggggtgc gggctggaca cgtactgtcc acctcggccg gcaacagtgc tcccaacgct
     2401 cccatggcca tgctacatat cagctccaac cccgagaaag agtttgatga tgtcattgac
     2461 aacattatgc gcctggacag cgtgctgggc tacatcaacc ctgagatgca gatgcctaac
     2521 acgctgcccc tgtctagcag ccacctgaac gtgtacagcg gtgaccccca ggtcacagcc
     2581 tccatggtgg gtgtcaccag cagcgcctgc cctgccgacc tgactcagaa gcgagagcta
     2641 acagatgctg agagcagagc cctggccaag gagcggcaga agaaagacaa tcacaaccta
     2701 attgagagaa gacgcaggtt caacatcaat gaccggatca aggagctggg aatgctgatc
     2761 cccaaggcca acgacctgga cgtgcgctgg aacaaaggca ccatcctcaa ggcctctgtg
     2821 gattacatcc ggaggatgca gaaggacctg cagaagtccc gggagctgga gaaccactcc
     2881 cggcgcctgg agatgactaa caagcagctc tggctccgca tccaggagct ggagatgcag
     2941 gcacgcgtgc acggcctccc caccacctcg ccgtcgggtg tgaatatggc cgagctggcc
     3001 cagcaggtgg tgaagcaaga gttgcccagt gaggatggcc caggggaggc gctgatgctg
     3061 gggcctgagg tccctgagcc tgagcaaatg ccggctcttc ctccccaggc tccgctgccc
     3121 tcggccgccc agccacagtc tccgttccat cacctggact tcagccatgg cctgagcttt
     3181 gggggtgggg gcgacgaggg gcccacaggt taccccgata ccctggggac agagcacggc
     3241 tccccattcc ccaacctgtc caagaaggat ctggacttaa tgctcctaga tgactccctg
     3301 ctccccctgg cctctgaccc cctcttttct accatgtctc ctgaggcctc caaggccagc
     3361 agccgccgga gcagcttcag catggaggag ggtgatgttc tgtgacaatt ggatccgatc
     3421 tagatcgagt taattaagaa ttccgccccc cccccctctc cctccccccc ccctaacgtt
     3481 actggccgaa gccgcttgga ataaggccgg tgtgcgtttg tctatatgtt attttccacc
     3541 atattgccgt cttttggcaa tgtgagggcc cggaaacctg gccctgtctt cttgacgagc
     3601 attcctaggg gtctttcccc tctcgccaaa ggaatgcaag gtctgttgaa tgtcgtgaag
     3661 gaagcagttc ctctggaagc ttcttgaaga caaacaacgt ctgtagcgac cctttgcagg
     3721 cagcggaacc ccccacctgg cgacaggtgc ctctgcggcc aaaagccacg tgtataagat
     3781 acacctgcaa aggcggcaca accccagtgc cacgttgtga gttggatagt tgtggaaaga
     3841 gtcaaatggc tctcctcaag cgtattcaac aaggggctga aggatgccca gaaggtaccc
     3901 cattgtatgg gatctgatct ggggcctcgg tacacatgct ttacatgtgt ttagtcgagg
     3961 ttaaaaaaac gtctaggccc cccgaaccac ggggacgtgg ttttcctttg aaaaacacga
     4021 tgataatatg gccacaacca tgaccgagta caagcccacg gtgcgcctcg ccacccgcga
     4081 cgacgtcccc agggccgtac gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg
     4141 ccacaccgtc gatccggacc gccacatcga gcgggtcacc gagctgcaag aactcttcct
     4201 cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg ccgcggtggc
     4261 ggtctggacc acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg
     4321 catggccgag ttgagcggtt cccggctggc cgcgcagcaa cagatggaag gcctcctggc
     4381 gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc gtcggcgtct cgcccgacca
     4441 ccagggcaag ggtctgggca gcgccgtcgt gctccccgga gtggaggcgg ccgagcgcgc
     4501 cggggtgccc gccttcctgg agacctccgc gccccgcaac ctccccttct acgagcggct
     4561 cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac
     4621 ccgcaagccc ggtgcctgac gcccgcccca cgacccgcag cgcccgaccg aaaggagcgc
     4681 acgaccccat gcatcgatga tctagagctc gctgatcagc ctcgactgtg ccttctagtt
     4741 gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc
     4801 ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt
     4861 ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca
     4921 ggcatgctgg ggatgcggtg ggctctatct gcggccgcca ccgcggtgga gctccagctt
     4981 ttgttccctt tagtgagggt taattgcgcg cttggcgtaa tcatggtcat agctgtttcc
     5041 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     5101 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     5161 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     5221 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     5281 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     5341 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     5401 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     5461 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     5521 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     5581 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     5641 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     5701 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     5761 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     5821 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     5881 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     5941 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     6001 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     6061 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     6121 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     6181 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     6241 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     6301 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     6361 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     6421 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     6481 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     6541 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     6601 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     6661 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     6721 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     6781 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     6841 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     6901 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     6961 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     7021 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     7081 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     7141 aggggttccg cgcacatttc cccgaaaagt gccacctgac gcgccctgta gcggcgcatt
     7201 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     7261 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     7321 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     7381 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     7441 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     7501 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     7561 ctattggtta aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat
     7621 taacgcttac aatttccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt
     7681 gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa ggcgattaag
     7741 ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca gtgagcgcgc
     7801 gtaatacgac tcactatagg gcgaattggg taccgggccc cccctcgagg tc
//
