LOCUS       sequence-354454-        5408 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5408)
  AUTHORS   Koivusalo M, Welch C, Hayashi H, Scott CC, Kim M, Alexander T, 
            Touret N, Hahn KM, Grinstein S
  TITLE     Amiloride inhibits macropinocytosis by lowering submembranous pH and
            preventing Rac1 and Cdc42 signaling.
  JOURNAL   J Cell Biol. 2010 Feb 22;188(4):547-63. Epub 2010 Feb 15.
  PUBMED    20156964
REFERENCE   2  (bases 1 to 5408)
  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: "J Cell 
            Biol."; date: "2010-02-22"; volume: "188(4)"; pages: "547-63. Epub 
            2010 Feb 15"
COMMENT     Sequence Label: mCherry-SEpHluorin
FEATURES             Location/Qualifiers
     source          1..5408
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        100..291
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     248..268
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        complement(293..397)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      424..879
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(511..530)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     721..742
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    886..1007
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(897..916)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     951..970
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1130..1846)
                     /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(1173..1193)
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     1788..1816
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     CDS             complement(1859..2566)
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(2134..2153)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2403..2421
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     promoter        complement(2635..2838)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(2664..2684)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        2839..3142
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     rep_origin      complement(3317..3905)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3397..3416)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    4234..4281
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     4306..4325
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     CDS             complement(4513..5307)
                     /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             complement(4513..5307)
                     /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(4624..4643)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     5234..5253
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(join(5342..5408,1..291))
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      join(5356..5408,1..83)
                     /label=SV40 ori
                     /note="SV40 origin of replication"
ORIGIN
        1 gcctcggcct ctgcataaat aaaaaaaatt agtcagccat ggggcggaga atgggcggaa
       61 ctgggcggag ttaggggcgg gatgggcgga gttaggggcg ggactatggt tgctgactaa
      121 ttgagatgca tgctttgcat acttctgcct gctggggagc ctggggactt tccacacctg
      181 gttgctgact aattgagatg catgctttgc atacttctgc ctgctgggga gcctggggac
      241 tttccacacc ctaactgaca cacattccac agctggttct ttccgcctca ggactcttcc
      301 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
      361 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
      421 ctgacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
      481 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
      541 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
      601 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
      661 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
      721 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
      781 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
      841 ttaacgcgaa ttttaacaaa atattaacgc ttacaattta cgccttaaga tacattgatg
      901 agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg
      961 atgctattgc tttatttgta accattataa gctgcaataa acaagttaac aacaacaatt
     1021 gcattcattt tatgtttcag gttcaggggg aggtgtggga ggttttttaa agcaagtaaa
     1081 acctctacaa atgtggtatg gctgattatg atctagagtc gcggccgctt tatttgtata
     1141 gttcatccat gccatgtgta atcccagcag ctgttacaaa ctcaagaagg accatgtggt
     1201 ctctcttttc gttgggatct ttcgaaagag tagaagttgt aaacaggtaa tggttgtctg
     1261 gtaaaaggac gggcccatcg ccaattggag tattttgttg ataatggtct gctagttgaa
     1321 cgcctccatc ttcaatgttg tgtctaattt tgaagttagc tttgattcca ttcttttgtt
     1381 tgtctgccat gatgtacacc tggtgatcgt tatagttgta ttccaatttg tgtccaagaa
     1441 tgtttccatc ttctttaaaa tcaatacctt ttaactcgat tctattaaca agggtatcac
     1501 cttcaaactt gacttcagca cgtgtcttgt agttcccgtc atctttgaaa aatatagttc
     1561 tttcctgtac ataaccttcg ggcatggcac tcttgaaaaa gtcatgccgt ttcatatgat
     1621 ctgggtatct tgaaaagcat tgaacaccat aagttaaagt agtgacaagt gttggccaag
     1681 gaacaggtag ttttccagta gtgcaaataa atttaagggt aagttttccg tatgttgcat
     1741 caccttcacc ctctccactg acagaaaatt tgtgcccatt aacatcacca tctaattcaa
     1801 caagaattgg gacaactcca gtgaaaagtt cttctccttt actcatggtg gcggatccct
     1861 tgtacagctc gtccatgccg ccggtggagt ggcggccctc ggcgcgttcg tactgttcca
     1921 cgatggtgta gtcctcgttg tgggaggtga tgtccaactt gatgttgacg ttgtaggcgc
     1981 cgggcagctg cacgggcttc ttggccttgt aggtggtctt gacctcagcg tcgtagtggc
     2041 cgccgtcctt cagcttcagc ctctgcttga tctcgccctt cagggcgccg tcctcggggt
     2101 acatccgctc ggaggaggcc tcccagccca tggtcttctt ctgcattacg gggccgtcgg
     2161 aggggaagtt ggtgccgcgc agcttcacct tgtagatgaa ctcgccgtcc tgcagggagg
     2221 agtcctgggt cacggtcacc acgccgccgt cctcgaagtt catcacgcgc tcccacttga
     2281 agccctcggg gaaggacagc ttcaagtagt cggggatgtc ggcggggtgc ttcacgtagg
     2341 ccttggagcc gtacatgaac tgaggggaca ggatgtccca ggcgaagggc agggggccac
     2401 ccttggtcac cttcagcttg gcggtctggg tgccctcgta ggggcggccc tcgccctcgc
     2461 cctcgatctc gaactcgtgg ccgttcacgg agccctccat gtgcaccttg aagcgcatga
     2521 actccttgat gatggccatg ttatcctcct cgcccttgct caccatggga attcgaagct
     2581 tgagctcgag atctgagtcc ggtagcgcta gcggatctga cggttcacta aaccagctct
     2641 gcttatatag acctcccacc gtacacgcct accgcccatt tgcgtcaatg gggcggagtt
     2701 gttacgacat tttggaaagt cccgttgatt ttggtgccaa aacaaactcc cattgacgtc
     2761 aatggggtgg agacttggaa atccccgtga gtcaaaccgc tatccacgcc cattgatgta
     2821 ctgccaaaac cgcatcacca tggtaatagc gatgactaat acgtagatgt actgccaagt
     2881 aggaaagtcc cataaggtca tgtactgggc ataatgccag gcgggccatt taccgtcatt
     2941 gacgtcaata gggggcgtac ttggcatatg atacacttga tgtactgcca agtgggcagt
     3001 ttaccgtaaa tactccaccc attgacgtca atggaaagtc cctattggcg ttactatggg
     3061 aacatacgtc attattgacg tcaatgggcg ggggtcgttg ggcggtcagc caggcgggcc
     3121 atttaccgta agttatgtaa cgcggaactc catatatggg ctatgaacta atgaccccgt
     3181 aattgattac tattaataac taatgcatgg cggtaatacg gttatccaca gaatcagggg
     3241 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     3301 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     3361 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     3421 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     3481 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     3541 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     3601 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     3661 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     3721 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     3781 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     3841 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     3901 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     3961 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     4021 aaaaatgaag ttttaaatca atctaaagta tatatgagta acctgaggct atggcagggc
     4081 ctgccgcccc gacgttggct gcgagccctg ggccttcacc cgaacttggg gggtggggtg
     4141 gggaaaagga agaaacgcgg gcgtattggc cccaatgggg tctcggtggg gtatcgacag
     4201 agtgccagcc ctgggaccga accccgcgtt tatgaacaaa cgacccaaca ccgtgcgttt
     4261 tattctgtct ttttattgcc gtcatagcgc gggttccttc cggtattgtc tccttccgtg
     4321 tttcagttag cctcccccta gggtgggcga agaactccag catgagatcc ccgcgctgga
     4381 ggatcatcca gccggcgtcc cggaaaacga ttccgaagcc caacctttca tagaaggcgg
     4441 cggtggaatc gaaatctcgt gatggcaggt tgggcgtcgc ttggtcggtc atttcgaacc
     4501 ccagagtccc gctcagaaga actcgtcaag aaggcgatag aaggcgatgc gctgcgaatc
     4561 gggagcggcg ataccgtaaa gcacgaggaa gcggtcagcc cattcgccgc caagctcttc
     4621 agcaatatca cgggtagcca acgctatgtc ctgatagcgg tccgccacac ccagccggcc
     4681 acagtcgatg aatccagaaa agcggccatt ttccaccatg atattcggca agcaggcatc
     4741 gccatgggtc acgacgagat cctcgccgtc gggcatgctc gccttgagcc tggcgaacag
     4801 ttcggctggc gcgagcccct gatgctcttc gtccagatca tcctgatcga caagaccggc
     4861 ttccatccga gtacgtgctc gctcgatgcg atgtttcgct tggtggtcga atgggcaggt
     4921 agccggatca agcgtatgca gccgccgcat tgcatcagcc atgatggata ctttctcggc
     4981 aggagcaagg tgagatgaca ggagatcctg ccccggcact tcgcccaata gcagccagtc
     5041 ccttcccgct tcagtgacaa cgtcgagcac agctgcgcaa ggaacgcccg tcgtggccag
     5101 ccacgatagc cgcgctgcct cgtcttgcag ttcattcagg gcaccggaca ggtcggtctt
     5161 gacaaaaaga accgggcgcc cctgcgctga cagccggaac acggcggcat cagagcagcc
     5221 gattgtctgt tgtgcccagt catagccgaa tagcctctcc acccaagcgg ccggagaacc
     5281 tgcgtgcaat ccatcttgtt caatcatgcg aaacgatcct catcctgtct cttgatcgat
     5341 ctttgcaaaa gcctaggcct ccaaaaaagc ctcctcacta cttctggaat agctcagagg
     5401 ccgaggcg
//
