LOCUS       sequence-350793-        5116 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of CD63-pHluorin for visualization of multivesicular 
            body-plasma membrane fusion..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5116)
  AUTHORS   Verweij FJ, Bebelman MP, Jimenez CR, Garcia-Vallejo JJ, Janssen H, 
            Neefjes J, Knol JC, de Goeij-de Haas R, Piersma SR, Baglio SR, 
            Verhage M, Middeldorp JM, Zomer A, van Rheenen J, Coppolino MG, 
            Hurbain I, Raposo G, Smit MJ, Toonen RFG, van Niel G, Pegtel DM
  TITLE     Quantifying exosome secretion from single cells reveals a modulatory
            role for GPCR signaling.
  JOURNAL   J Cell Biol. 2018 Mar 5;217(3):1129-1142. doi: 
            10.1083/jcb.201703206. Epub 2018 Jan 16.
  PUBMED    29339438
REFERENCE   2  (bases 1 to 5116)
  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: 
            "10.1083/jcb.201703206"; journalName: "J Cell Biol"; date: 
            "2018-03-5- 5"; volume: "217"; issue: "3"; pages: "1129-1142"
COMMENT     Sequence Label: pCMV-Sport6-CD63-pHluorin
FEATURES             Location/Qualifiers
     source          1..5116
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     89..108
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     342..359
                     /label=L4440
                     /note="L4440 vector, forward primer"
     polyA_signal    429..563
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(453..472)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     507..526
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     953..975
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     967..984
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     968..984
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     995..1014
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        995..1013
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1015..1039
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(1710..2426)
                     /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="MGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNDHQVYIMADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLFTTSTLSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(1750..1770)
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     2365..2393
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     regulatory      2423..2432
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(2569..2593)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        complement(2594..2612)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2595..2612)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2623..2639)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2623..2639)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2744..2768)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        complement(2765..2968)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(2794..2814)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        2969..3272
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     CDS             join(3643..3711,3712..4503)
                     /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     complement(3861..3880)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     protein_bind    4605..4638
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     rep_origin      join(4716..5116,1..188)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
       61 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
      121 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
      181 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
      241 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
      301 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
      361 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
      421 gcaggatcga tccagacatg ataagataca ttgatgagtt tggacaaacc acaactagaa
      481 tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta tttgtaacca
      541 ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg tttcaggttc
      601 agggggaggt gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt ggtatggctg
      661 attatgatca tgaacagact gtgaggactg aggggcctga aatgagcctt gggactgtga
      721 atctaaaata cacaaacaat tagaatcact agctcctgtg tataatattt tcataaatca
      781 tactcagtaa gcaaaactct caagcagcaa gcatatgcag ctagtttaac acattataca
      841 cttaaaaatt ttatatttac cttagagctt taaatctctg taggtagttt gtccaattat
      901 gtcacaccac agaagtaagg ttccttcaca aagatcccaa gctagcagtt ttcccagtca
      961 cgacgttgta aaacgacggc cagtgcctag cttataatac gactcactat agggaccact
     1021 ttgtacaaga aagctgggta cgcgtaagct tgggcccctc gagggatact ctagacctac
     1081 atcacctcgt agccacttct gatactcttc acgaggcagc aggcaaagac aattcccaaa
     1141 acctcgacaa aagcaattcc aagggctgct gcagctacca ccagcacatt tttcctcagc
     1201 cagcccccaa tcttctccac acagccctcc ttatggatcg ccttctcgtt gaaattaatc
     1261 ccacagccca cagtaacatt aatgcagcag gagtcgggga ctcggttctt cgacatggaa
     1321 gggattttct cccaatctgt gtagttagca gccccacagc acttaaaatc tgcctgcatc
     1381 ctgtccagga tcgaagcagt gtggttgttt ttcgggtaat tctccatctg ctgccggaag
     1441 ttgttattaa actctgacat caccttatct ctaaacacat agccagcaat ggctgcggcc
     1501 acctccacca acatgataag agacagaaag atggcaaacg tgatcataag acaatagttc
     1561 tccttgcagg ccccgcagca gcccacaaaa gccaccagga agaggaagac acccactgcg
     1621 atgatgacca ctggcaacag agagccaggg gtagccccct ggattatggt ctgactcagg
     1681 acaagagatc tgattcgagc tccaccggtt ttgtatagtt catccatgcc atgtgtaatc
     1741 ccagcagctg ttacaaactc aagaaggacc atgtggtctc tcttttcgtt gggatctttc
     1801 gaaagagtag aagttgtaaa caggtaatgg ttgtctggta aaaggacggg cccatcgcca
     1861 attggagtat tttgttgata atggtctgct agttgaacgc ctccatcttc aatgttgtgt
     1921 ctaattttga agttagcttt gattccattc ttttgtttgt ctgccatgat gtacacctgg
     1981 tgatcgttat agttgtattc caatttgtgt ccaagaatgt ttccatcttc tttaaaatca
     2041 atacctttta actcgattct attaacaagg gtatcacctt caaacttgac ttcagcacgt
     2101 gtcttgtagt tcccgtcatc tttgaaaaat atagttcttt cctgtacata accttcgggc
     2161 atggcactct tgaaaaagtc atgccgtttc atatgatctg ggtatcttga aaagcattga
     2221 acaccataag ttaaagtagt gacaagtgtt ggccaaggaa caggtagttt tccagtagtg
     2281 caaataaatt taagggtaag ttttccgtat gttgcatcac cttcaccctc tccactgaca
     2341 gaaaatttgt gcccattaac atcaccatct aattcaacaa gaattgggac aactccagtg
     2401 aaaagttctt ctcctttact tcccatggtg gctagagatc tctgtgcccc gacacccacg
     2461 gcaatcagtc ccactgcaca ggcgcaaaag gccagcagga ggacgtagag caagaacttc
     2521 acacatttca ttcctccttc caccgccatg gaattccgga ccggtaccag cctgcttttt
     2581 tgtacaaact tgttctatag tgtcacctaa ataggcctaa tggtcatagc tgtttcctgt
     2641 gtgaaattgt tatccgtccc gcggcctagg ctagagtccg gaggctggat cggtcccggt
     2701 gtcttctatg gaggtcaaaa cagcgtggat ggcgtctcca ggcgatctga cggttcacta
     2761 aacgagctct gcttatatag acctcccacc gtacacgcct accgcccatt tgcgtcaatg
     2821 gggcggagtt gttacgacat tttggaaagt cccgttgatt ttggtgccaa aacaaactcc
     2881 cattgacgtc aatggggtgg agacttggaa atccccgtga gtcaaaccgc tatccacgcc
     2941 cattgatgta ctgccaaaac cgcatcacca tggtaatagc gatgactaat acgtagatgt
     3001 actgccaagt aggaaagtcc cataaggtca tgtactgggc ataatgccag gcgggccatt
     3061 taccgtcatt gacgtcaata gggggcgtac ttggcatatg atacacttga tgtactgcca
     3121 agtgggcagt ttaccgtaaa tactccaccc attgacgtca atggaaagtc cctattggcg
     3181 ttactatggg aacatacgtc attattgacg tcaatgggcg ggggtcgttg ggcggtcagc
     3241 caggcgggcc atttaccgta agttatgtaa cgacatgcat ctaatgcgtg aaagggcctc
     3301 gtaatacgcc tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt
     3361 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     3421 aatatgtatc cgctcatgcc aggtcttgga ctggtgagaa cggcttgctc ggcagcttcg
     3481 atgtgtgctg gagggagaat aaaggtctaa gatgtgcgat agagggaagt cgcattgaat
     3541 tatgtgctgt gtagggatcg ctggtatcaa atatgtgtgc ccacccctgg catgagacaa
     3601 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     3661 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     3721 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     3781 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     3841 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccggacaa
     3901 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     3961 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     4021 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     4081 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     4141 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     4201 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     4261 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     4321 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
     4381 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     4441 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     4501 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     4561 tttaaaagga tctaggtgaa gatccttttt gataatctca tgccataact tcgtataatg
     4621 tatgctatac gaagttatgg catgaccaaa atcccttaac gtgagttttc gttccactga
     4681 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     4741 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     4801 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     4861 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     4921 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     4981 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     5041 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     5101 cgtgagctat gagaaa
//
