LOCUS       sequence-172523-        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        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             603..1316
                     /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(633..661)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1256..1276
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             1335..2030
                     /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    2043..2108
                     /label=MCS
                     /note="multiple cloning site"
     polyA_signal    2232..2353
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2269..2288)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2323..2342
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2360..2815)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2497..2518)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2709..2728
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2842..2946
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2948..3305
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2948..3139
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2971..2991)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3156..3291
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3340..4134
                     /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             3340..4134
                     /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(3394..3413)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4004..4023
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4322..4341)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4366..4413
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4742..5330
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5231..5250
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcca
      601 ccatgagtaa aggagaagaa cttttcactg gagttgtccc aattcttgtt gaattagatg
      661 gtgatgttaa tgggcacaaa ttttctgtca gtggagaggg tgaaggtgat gcaacatacg
      721 gaaaacttac ccttaaattt atttgcacta ctggaaaact acctgttcca tggccaacac
      781 ttgtcactac gttaacttat ggtgttcaat gcttttcaag atacccagat catatgaaac
      841 ggcatgactt tttcaagagt gccatgcccg aaggttacgt acaggaaaga actatatttt
      901 tcaaagatga cgggaactac aagacacgtg ctgaagtcaa gtttgaaggt gatacccttg
      961 ttaatagaat cgagttaaaa ggtattgatt ttaaagaaga tggaaacatt cttggacaca
     1021 aattggaata caactataac gatcaccagg tgtacatcat ggcagacaaa caaaagaatg
     1081 gaatcaaagc taacttcaaa attagacaca acattgaaga tggaggcgtt caactagcag
     1141 accattatca acaaaatact ccaattggcg atgggcccgt ccttttacca gacaaccatt
     1201 acctgtttac aacttctact ctttcgaaag atcccaacga aaagagagac cacatggtcc
     1261 ttcttgagtt tgtaacagct gctgggatta cacatggcat ggatgaacta tacaaaggtg
     1321 gaggaggtag atccatggtg agcgagctga ttaaggagaa catgcacatg aagctgtaca
     1381 tggagggcac cgtgaacaac caccacttca agtgcacatc cgagggcgaa ggcaagccct
     1441 acgagggcac ccagaccatg agaatcaagg cggtcgaggg cggccctctc cccttcgcct
     1501 tcgacatcct ggctaccagc ttcatgtacg gcagcaaaac cttcatcaac cacacccagg
     1561 gcatccccga cttctttaag cagtccttcc ccgagggctt cacatgggag agagtcacca
     1621 catacgaaga cgggggcgtg ctgaccgcta cccaggacac cagcctccag gacggctgcc
     1681 tcatctacaa cgtcaagatc agaggggtga acttcccatc caacggccct gtgatgcaga
     1741 agaaaacact cggctgggag gcctccaccg agaccctgta ccccgctgac ggcggcctgg
     1801 aaggcagagc cgacatggcc ctgaagctcg tgggcggggg ccacctgatc tgcaacttga
     1861 agaccacata cagatccaag aaacccgcta agaacctcaa gatgcccggc gtctactatg
     1921 tggacagaag actggaaaga atcaaggagg ccgacaaaga gacctacgtc gagcagcacg
     1981 aggtggctgt ggccagatac tgcgacctcc ctagcaaact ggggcacaga ggtggaggag
     2041 gttccggact cagatctcga gctcaagctt cgaattctgc agtcgacggt accgcgggcc
     2101 cgggatccac cggatctaga taactgatca taatcagcca taccacattt gtagaggttt
     2161 tacttgcttt aaaaaacctc ccacacctcc ccctgaacct gaaacataaa atgaatgcaa
     2221 ttgttgttgt taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     2281 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     2341 tcaatgtatc ttaacgcgta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat
     2401 ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa
     2461 tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta
     2521 ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca
     2581 ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat
     2641 cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg
     2701 agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc
     2761 acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcaggt
     2821 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     2881 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     2941 aagagtcctg aggcggaaag aaccagctgt ggaatgtgtg tcagttaggg tgtggaaagt
     3001 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     3061 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     3121 agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
     3181 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
     3241 cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt
     3301 gcaaagatcg atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg
     3361 cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag
     3421 acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt
     3481 tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aagacgaggc agcgcggcta
     3541 tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg
     3601 ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt
     3661 gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat
     3721 ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg
     3781 atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca
     3841 gccgaactgt tcgccaggct caaggcgagc atgcccgacg gcgaggatct cgtcgtgacc
     3901 catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc
     3961 gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat
     4021 attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc
     4081 gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga
     4141 ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt
     4201 ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga
     4261 tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccctagg gggaggctaa
     4321 ctgaaacacg gaaggagaca ataccggaag gaacccgcgc tatgacggca ataaaaagac
     4381 agaataaaac gcacggtgtt gggtcgtttg ttcataaacg cggggttcgg tcccagggct
     4441 ggcactctgt cgatacccca ccgagacccc attggggcca atacgcccgc gtttcttcct
     4501 tttccccacc ccacccccca agttcgggtg aaggcccagg gctcgcagcc aacgtcgggg
     4561 cggcaggccc tgccatagcc tcaggttact catatatact ttagattgat ttaaaacttc
     4621 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     4681 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     4741 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     4801 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     4861 tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact
     4921 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     4981 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     5041 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     5101 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     5161 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     5221 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     5281 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     5341 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     5401 cgttatcccc tgattctgtg gataaccgta ttaccgccat gcat
//
