LOCUS       sequence-323903-        7501 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express pHluorin-mKate2-hLC3 (PK-hLC3) in mammalian cells for 
            monitoring autophagy.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7501)
  AUTHORS   Tanida I, Ueno T, Uchiyama Y
  TITLE     A Super-Ecliptic, pHluorin-mKate2, Tandem Fluorescent Protein-Tagged
            Human LC3 for the Monitoring of Mammalian Autophagy.
  JOURNAL   PLoS One. 2014 Oct 23;9(10):e110600. doi: 
            10.1371/journal.pone.0110600. eCollection 2014.
  PUBMED    25340751
REFERENCE   2  (bases 1 to 7501)
  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: "PLoS One.";
            date: "2014-10-23"; volume: "9(10):e110600. doi"; pages: " 
            10.1371/journal.pone.0110600. eCollection 2014"
COMMENT     Sequence Label: pEX-PK-hLC3
FEATURES             Location/Qualifiers
     source          1..7501
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    94..142
                     /note="synthetic polyadenylation signal"
     CDS             complement(167..766)
                     /codon_start=1
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     promoter        complement(797..1154)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      811..946
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(865..884)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     enhancer        963..1154
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     1111..1131
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     enhancer        1250..1629
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          1854..2870
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2794..2816
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2878..2897
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             2954..3667
                     /codon_start=1
                     /product="pH-sensitive mutant of green fluorescent protein 
                     (Ng et al., 2002)"
                     /label=superecliptic pHluorin
                     /note="excited strongly by blue light at neutral pH but 
                     minimally fluorescent at acidic pH"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNDHQVYIMADKQKNGIKA
                     NFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLFTTSTLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(2984..3012)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     3607..3627
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             3686..4381
                     /codon_start=1
                     /product="monomeric far-red fluorescent protein (Shcherbo 
                     et al., 2009)"
                     /label=mKate2
                     /note="mammalian codon-optimized"
                     /translation="MVSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIK
                     AVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLT
                     ATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMAL
                     KLVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARY
                     CDLPSKLGHR"
     CDS             4409..4783
                     /codon_start=1
                     /gene="MAP1LC3B"
                     /product="human LC3 protein"
                     /label=LC3B
                     /note="Atg8 family member found on autophagosomes"
                     /translation="MPSEKTFKQRRTFEQRVEDVRLIREQHPTKIPVIIERYKGEKQLP
                     VLDKTKFLVPDHVNMSELIKIIRRRLQLNANQAFFLLVNGHSMVSVSTPISEVYESEKD
                     EDGFLYMVYASQETFGMKLSV"
     primer_bind     complement(4886..4905)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4951..5006
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(5005..5024)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(5394..5413)
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     polyA_signal    5467..5515
                     /note="synthetic polyadenylation signal"
     CDS             join(5620..5688,5689..6480)
                     /codon_start=1
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      6680..7268
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7169..7188
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7422..7439
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gtgcggcgac gatagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc
       61 tcaagggcat cggtcgacag catctagttc gaacacacaa aaaaccaaca cacagatgta
      121 atgaaaataa agatatttta ttgcggccat gaccggttct agatcattag gcgccaggtt
      181 ttcgtgtcat acaccaggtc ctaggtccct cgggcacctc gacatcggcg gtcacggtga
      241 agcccaggcg ctcgtagaat ggaaggtttc ttggtgcgct tgtttcgaga aaggcgggca
      301 caccggcgcg ttcggcggcc tctacgccgg gcaacacgac ggcgctgccc aagcccttgc
      361 cctggtggtc ggggctcact cctacagtgg ccagaaacca ggcaggctcc ttgggacgat
      421 ggggggcaag caggccctcc atttgctgct gggcggccag gcggctgccg ctcagctcgg
      481 ccattcgggg gccgatctcg gcgaacacgg cgccggcctc aacgctctcg ggggtagtcc
      541 acacggccac ggccgcgccg tcgtcggcta cccacacctt gccgatgtcc agcccgacgc
      601 gggtcaggaa cagctcctgc aactctgtca cacgctcgat gtgccggtcg gggtccacgg
      661 tgtgccgggt agcggggtag tcggcgaaag cggcggccag agtgcggacg gcgcggggca
      721 catcatcgcg agtggccagg cgcacggtag gcttgtactc ggtcatggtg gcgctagtgt
      781 cagaagaatc gagctttttg caaaagccta ggcctccaaa aaagcctcct cactacttct
      841 ggaatagctc agaggcagag gcggcctcgg cctctgcata aataaaaaaa attagtcagc
      901 catggggcgg agaatgggcg gaactgggcg gagttagggg cgggatgggc ggagttaggg
      961 gcgggactat ggttgctgac taattgagat gcatgctttg catacttctg cctgctgggg
     1021 agcctgggga ctttccacac ctggttgctg actaattgag atgcatgctt tgcatacttc
     1081 tgcctgctgg ggagcctggg gactttccac accctaactg acacacattc cacagctggt
     1141 tctttccgcc tcagaaggta gctaaccaag ttcctctttc agaggttatt tcaggccatg
     1201 gtgctgcgca gatcagcgga agagcgccca atacgcaaac ggatccgtcg acattgatta
     1261 ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag
     1321 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
     1381 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
     1441 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
     1501 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
     1561 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
     1621 attaccatgg tcgaggtgag ccccacgttc tgcttcactc tccccatctc ccccccctcc
     1681 ccacccccaa ttttgtattt atttattttt taattatttt gtgcagcgat gggggcgggg
     1741 sgaggcggag aggtgcggcg gcagccaatc agagcggcgc gctccgaaag tttcctttta
     1801 tggcgaggcg gcggcggcgg cggccctata aaaagcgaag cgcgcggcgg gcgggagtcg
     1861 ctgcgcgctg ccttcgcccc gtgccccgct ccgccgccgc ctcgcgccgc ccgccccggc
     1921 tctgactgac cgcgttactc ccacaggtga gcgggcggga cggcccttct cctccgggct
     1981 gtaattagcg cttggtttaa tgacggcttg tttcttttct gtggctgcgt gaaagccttg
     2041 aggggctccg ggagggccct ttgtgcgggg ggagcggctc ggggggtgcg tgcgtgtgtg
     2101 tgtgcgtggg gagcgccgcg tgcggctccg cgctgcccgg cggctgtgag cgctgcgggc
     2161 gcggcgcggg gctttgtgcg ctccgcagtg tgcgcgaggg gagcgcggcc gggggcggtg
     2221 ccccgcggtg cggggggggc tgcgagggga acaaaggctg cgtgcggggt gtgtgcgtgg
     2281 gggggtgagc agggggtgtg ggcgcgtcgg tcgggctgca accccccctg cacccccctc
     2341 cccgagttgc tgagcacggc ccggcttcgg gtgcggggct ccgtacgggg cgtggcgcgg
     2401 ggctcgccgt gccgggcggg gggtggcggc aggtgggggt gccgggcggg gcggggccgc
     2461 ctcgggccgg ggagggctcg ggggaggggc gcggcggccc ccggagcgcc ggcggctgtc
     2521 gaggcgcggc gagccgcagc cattgccttt tatggtaatc gtgcgagagg gcgcagggac
     2581 ttcctttgtc ccaaatctgt gcggagccga aatctgggag gcgccgccgc accccctcta
     2641 gcgggcgcgg ggcgaagcgg tgcggcgccg gcaggaagga aatgggcggg gagggccttc
     2701 gtgcgtcgcc gcgccgccgt ccccttctcc ctctccagcc tcggggctgt ccgcgggggg
     2761 acggctgcct tcggggggga cggggcaggg cggggttcgg cttctggcgt gtgaccggcg
     2821 gctctagagc ctctgctaac catgttcatg ccttcttctt tttcctacag ctcctgggca
     2881 acgtgctggt tattgtgctg tctcatcatt ttggcaaaga attcttaagg gtaccgatat
     2941 cgctagcgcc accatgagta aaggagaaga acttttcact ggagttgtcc caattcttgt
     3001 tgaattagat ggtgatgtta atgggcacaa attttctgtc agtggagagg gtgaaggtga
     3061 tgcaacatac ggaaaactta cccttaaatt tatttgcact actggaaaac tacctgttcc
     3121 atggccaaca cttgtcacta cgttaactta tggtgttcaa tgcttttcaa gatacccaga
     3181 tcatatgaaa cggcatgact ttttcaagag tgccatgccc gaaggttacg tacaggaaag
     3241 aactatattt ttcaaagatg acgggaacta caagacacgt gctgaagtca agtttgaagg
     3301 tgataccctt gttaatagaa tcgagttaaa aggtattgat tttaaagaag atggaaacat
     3361 tcttggacac aaattggaat acaactataa cgatcaccag gtgtacatca tggcagacaa
     3421 acaaaagaat ggaatcaaag ctaacttcaa aattagacac aacattgaag atggaggcgt
     3481 tcaactagca gaccattatc aacaaaatac tccaattggc gatgggcccg tccttttacc
     3541 agacaaccat tacctgttta caacttctac tctttcgaaa gatcccaacg aaaagagaga
     3601 ccacatggtc cttcttgagt ttgtaacagc tgctgggatt acacatggca tggatgaact
     3661 atacaaaggt ggaggaggta gatccatggt gagcgagctg attaaggaga acatgcacat
     3721 gaagctgtac atggagggca ccgtgaacaa ccaccacttc aagtgcacat ccgagggcga
     3781 aggcaagccc tacgagggca cccagaccat gagaatcaag gcggtcgagg gcggccctct
     3841 ccccttcgcc ttcgacatcc tggctaccag cttcatgtac ggcagcaaaa ccttcatcaa
     3901 ccacacccag ggcatccccg acttctttaa gcagtccttc cccgagggct tcacatggga
     3961 gagagtcacc acatacgaag acgggggcgt gctgaccgct acccaggaca ccagcctcca
     4021 ggacggctgc ctcatctaca acgtcaagat cagaggggtg aacttcccat ccaacggccc
     4081 tgtgatgcag aagaaaacac tcggctggga ggcctccacc gagaccctgt accccgctga
     4141 cggcggcctg gaaggcagag ccgacatggc cctgaagctc gtgggcgggg gccacctgat
     4201 ctgcaacttg aagaccacat acagatccaa gaaacccgct aagaacctca agatgcccgg
     4261 cgtctactat gtggacagaa gactggaaag aatcaaggag gccgacaaag agacctacgt
     4321 cgagcagcac gaggtggctg tggccagata ctgcgacctc cctagcaaac tggggcacag
     4381 aggtggagga ggttccggac tcagatctat gccgtcggag aagaccttca agcagcgccg
     4441 caccttcgaa caaagagtag aagatgtccg acttattcga gagcagcatc caaccaaaat
     4501 cccggtgata atagaacgat acaagggtga gaagcagctt cctgttctgg ataaaacaaa
     4561 gttccttgta cctgaccatg tcaacatgag tgagctcatc aagataatta gaaggcgctt
     4621 acagctcaat gctaatcagg ccttcttcct gttggtgaac ggacacagca tggtcagcgt
     4681 ctccacacca atctcagagg tgtatgagag tgagaaagat gaagatggat tcctgtacat
     4741 ggtctacgcc tcccaggaga cgttcgggat gaaattgtca gtgtaagtcg agcccgggag
     4801 ctcaattcac tcctcaggtg caggctgcct atcagaaggt ggtggctggt gtggccaatg
     4861 ccctggctca caaataccac tgagatcttt ttccctctgc caaaaattat ggggacatca
     4921 tgaagcccct tgagcatctg acttctggct aataaaggaa atttattttc attgcaatag
     4981 tgtgttggaa ttttttgtgt ctctcactcg gaaggacata tgggagggca aatcatttaa
     5041 aacatcagaa tgagtatttg gtttagagtt tggcaacata tgcccatatg ctggctgcca
     5101 tgaacaaagg ttggctataa agaggtcatc agtatatgaa acagccccct gctgtccatt
     5161 ccttattcca tagaaaagcc ttgacttgag gttagatttt ttttatattt tgttttgtgt
     5221 tatttttttc tttaacatcc ctaaaatttt ccttacatgt tttactagcc agatttttcc
     5281 tcctctcctg actactccca gtcatagctg tccctcttct cttatggaga tccctcgacc
     5341 tgcagcccaa gcttggccgc cgaggccatg ttctggcacc tgcacttgca ctggggacag
     5401 cctattttgc tagtttgttt tgtttcgttt tgttttgatg gagagcgtat gttagtacta
     5461 tcgattcaca caaaaaacca acacacagat gtaatgaaaa taaagatatt ttattgcggc
     5521 ctgtccaata cctcccgtac cttaatatta cttacttatc cttgagagac gtactagtaa
     5581 ccctgataaa tgcttcaata atattgaaaa aggacgagta tgagcatcca acattttcgt
     5641 gtcgcactca ttcccttctt tgcggcattt tgcttgcctg tttttgcaca ccccgaaacg
     5701 ctggtgaaag taaaagatgc tgaagatcaa ctgggtgcaa gagtgggcta tatcgaactg
     5761 gatctcaata gcggcaagat ccttgagtct ttccgccccg aagaacgatt cccgatgatg
     5821 agcactttca aagtactgct atgtggcgcg gtgttgtccc gtatagacgc cgggcaagag
     5881 cagcttggtc gccgtataca ctactcacaa aacgacttgg ttgagtactc gccggtcacg
     5941 gaaaagcatc ttacggatgg catgacggta agagaattgt gtagtgctgc cattaccatg
     6001 agcgacaaca ccgcggccaa cttacttctg acaacgatcg gaggccctaa ggagctgact
     6061 gcatttcttc ataatatggg tgatcatgtg acccggcttg accgctggga accagagttg
     6121 aacgaagcca taccgaacga cgagcgtgat accacgatgc cagtagcaat ggccacaact
     6181 cttcggaaac tactcactgg cgaacttctt actctagcat cacgacagca gctcatagac
     6241 tggatggagg cggacaaagt agcaggacca cttcttcgct cggccctccc tgctggctgg
     6301 ttcattgctg acaaatcggg ggccggtgaa cgcggctctc gcggcatcat tgctgcgctg
     6361 gggcctgatg gtaagccctc acgaatcgta gtgatctaca cgacggggag tcaggccact
     6421 atggacgaac gaaataggca gatcgctgag atcggtgcct cactgatcaa gcactggtaa
     6481 ccactgcagt ggtttagcat ttgcggccgc tgtcagacca agtttactca tatatacttt
     6541 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     6601 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     6661 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     6721 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     6781 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     6841 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     6901 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     6961 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     7021 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     7081 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     7141 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     7201 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     7261 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     7321 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     7381 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     7441 aagcggaaga gcgctgccgg cacctgtcct acgagttgca tgataaagaa gacagtcata
     7501 a
//
