LOCUS       sequence-252335-        5444 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Backbone plasmid for expressing pHluorin-mKate2-fusion protein in 
            mammalan cells, which contains multi-cloning site like pEGFP-C1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5444)
  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 5444)
  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: pHmK-C1
FEATURES             Location/Qualifiers
     source          1..5444
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..304
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        305..508
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     459..479
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             543..1256
                     /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(573..601)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1196..1216
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             1275..1970
                     /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"
     misc_feature    1983..2048
                     /label=MCS
                     /note="multiple cloning site"
     polyA_signal    2172..2293
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2209..2228)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2263..2282
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2300..2755)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2437..2458)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2649..2668
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2782..2886
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2888..3245
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2888..3079
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2911..2931)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3096..3231
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3280..4074
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             3280..4074
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3334..3353)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3944..3963
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4262..4281)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4306..4353
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4682..5270
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5171..5190
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
       61 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      121 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      181 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      241 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      301 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      361 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      421 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      481 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcca
      541 ccatgagtaa aggagaagaa cttttcactg gagttgtccc aattcttgtt gaattagatg
      601 gtgatgttaa tgggcacaaa ttttctgtca gtggagaggg tgaaggtgat gcaacatacg
      661 gaaaacttac ccttaaattt atttgcacta ctggaaaact acctgttcca tggccaacac
      721 ttgtcactac gttaacttat ggtgttcaat gcttttcaag atacccagat catatgaaac
      781 ggcatgactt tttcaagagt gccatgcccg aaggttacgt acaggaaaga actatatttt
      841 tcaaagatga cgggaactac aagacacgtg ctgaagtcaa gtttgaaggt gatacccttg
      901 ttaatagaat cgagttaaaa ggtattgatt ttaaagaaga tggaaacatt cttggacaca
      961 aattggaata caactataac gatcaccagg tgtacatcat ggcagacaaa caaaagaatg
     1021 gaatcaaagc taacttcaaa attagacaca acattgaaga tggaggcgtt caactagcag
     1081 accattatca acaaaatact ccaattggcg atgggcccgt ccttttacca gacaaccatt
     1141 acctgtttac aacttctact ctttcgaaag atcccaacga aaagagagac cacatggtcc
     1201 ttcttgagtt tgtaacagct gctgggatta cacatggcat ggatgaacta tacaaaggtg
     1261 gaggaggtag atccatggtg agcgagctga ttaaggagaa catgcacatg aagctgtaca
     1321 tggagggcac cgtgaacaac caccacttca agtgcacatc cgagggcgaa ggcaagccct
     1381 acgagggcac ccagaccatg agaatcaagg cggtcgaggg cggccctctc cccttcgcct
     1441 tcgacatcct ggctaccagc ttcatgtacg gcagcaaaac cttcatcaac cacacccagg
     1501 gcatccccga cttctttaag cagtccttcc ccgagggctt cacatgggag agagtcacca
     1561 catacgaaga cgggggcgtg ctgaccgcta cccaggacac cagcctccag gacggctgcc
     1621 tcatctacaa cgtcaagatc agaggggtga acttcccatc caacggccct gtgatgcaga
     1681 agaaaacact cggctgggag gcctccaccg agaccctgta ccccgctgac ggcggcctgg
     1741 aaggcagagc cgacatggcc ctgaagctcg tgggcggggg ccacctgatc tgcaacttga
     1801 agaccacata cagatccaag aaacccgcta agaacctcaa gatgcccggc gtctactatg
     1861 tggacagaag actggaaaga atcaaggagg ccgacaaaga gacctacgtc gagcagcacg
     1921 aggtggctgt ggccagatac tgcgacctcc ctagcaaact ggggcacaga ggtggaggag
     1981 gttccggact cagatctcga gctcaagctt cgaattctgc agtcgacggt accgcgggcc
     2041 cgggatccac cggatctaga taactgatca taatcagcca taccacattt gtagaggttt
     2101 tacttgcttt aaaaaacctc ccacacctcc ccctgaacct gaaacataaa atgaatgcaa
     2161 ttgttgttgt taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     2221 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     2281 tcaatgtatc ttaacgcgta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat
     2341 ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa
     2401 tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta
     2461 ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca
     2521 ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat
     2581 cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg
     2641 agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc
     2701 acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcaggt
     2761 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     2821 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     2881 aagagtcctg aggcggaaag aaccagctgt ggaatgtgtg tcagttaggg tgtggaaagt
     2941 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     3001 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     3061 agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
     3121 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
     3181 cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt
     3241 gcaaagatcg atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg
     3301 cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag
     3361 acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt
     3421 tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aagacgaggc agcgcggcta
     3481 tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg
     3541 ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt
     3601 gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat
     3661 ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg
     3721 atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca
     3781 gccgaactgt tcgccaggct caaggcgagc atgcccgacg gcgaggatct cgtcgtgacc
     3841 catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc
     3901 gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat
     3961 attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc
     4021 gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga
     4081 ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt
     4141 ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga
     4201 tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccctagg gggaggctaa
     4261 ctgaaacacg gaaggagaca ataccggaag gaacccgcgc tatgacggca ataaaaagac
     4321 agaataaaac gcacggtgtt gggtcgtttg ttcataaacg cggggttcgg tcccagggct
     4381 ggcactctgt cgatacccca ccgagacccc attggggcca atacgcccgc gtttcttcct
     4441 tttccccacc ccacccccca agttcgggtg aaggcccagg gctcgcagcc aacgtcgggg
     4501 cggcaggccc tgccatagcc tcaggttact catatatact ttagattgat ttaaaacttc
     4561 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     4621 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     4681 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     4741 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     4801 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     4861 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     4921 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     4981 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     5041 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     5101 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     5161 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     5221 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     5281 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     5341 cgttatcccc tgattctgtg gataaccgta ttaccgccat gcattagtta ttaatagtaa
     5401 tcaattacgg ggtcattagt tcatagccca tatatggagt tccg
//
