LOCUS       sequence-350176-        7055 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of tetraspanin CD63 fused to the MS2 dimer and 
            C-terminal HA tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7055)
  AUTHORS   Hung ME, Leonard JN
  TITLE     A platform for actively loading cargo RNA to elucidate limiting 
            steps in EV-mediated delivery.
  JOURNAL   J Extracell Vesicles. 2016 May 13;5:31027. doi: 
            10.3402/jev.v5.31027. eCollection 2016.
  PUBMED    27189348
REFERENCE   2  (bases 1 to 7055)
  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 Extracell
            Vesicles."; date: "2016-05-13"; volume: "5:31027. doi"; pages: " 
            10.3402/jev.v5.31027. eCollection 2016"
COMMENT     Sequence Label: pMEH343 CD63 MS2 HA
FEATURES             Location/Qualifiers
     source          1..7055
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        90..469
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        470..673
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     624..644
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     718..737
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        718..736
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             756..1469
                     /codon_start=1
                     /product="human CD63 tetraspanin"
                     /label=CD63
                     /note="enriched in intraluminal vesicles of late endosomes 
                     (multivesicular bodies) and in exosomes"
                     /translation="MAVEGGMKCVKFLLYVLLLAFCACAVGLIAVGVGAQLVLSQTIIQ
                     GATPGSLLPVVIIAVGVFLFLVAFVGCCGACKENYCLMITFAIFLSLIMLVEVAAAIAG
                     YVFRDKVMSEFNNNFRQQMENYPKNNHTASILDRMQADFKCCGAANYTDWEKIPSMSKN
                     RVPDSCCINVTVGCGINFNEKAIHKEGCVEKIGGWLRKNVLVVAAAALGIAFVEVLGIV
                     FACCLVKSIRSGYEVM"
     CDS             2205..2231
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(2332..2349)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2338..2562
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2608..3036
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2695..2714)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2905..2926
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3045..3065)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3050..3379
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3230..3365
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3292..3311
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3428..4453
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             3428..4453
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     polyA_signal    4583..4704
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4620..4639)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4674..4693
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4753..4769)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4753..4769)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4766..4788)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4777..4793
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(4801..4807,4808..4825,4826..4831))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4846..4867
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4984..5001)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5155..5743)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5235..5254)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5914..6705,6706..6774))
                     /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     6537..6556
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6775..6879)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6954..6973)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gcaaggcttg accgacaatt gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct
       61 tcgcgatgta cgggccagat atacgcgttg acattgatta ttgactagtt attaatagta
      121 atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac
      181 ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac
      241 gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt
      301 acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat
      361 tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga
      421 ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt
      481 ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca
      541 ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg
      601 tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta
      661 tataagcaga gctctctggc taactagaga acccactgct tactggctta tcgaaattaa
      721 tacgactcac tatagggaga cccaagctgg gtaccatggc ggtggaagga ggaatgaaat
      781 gtgtgaagtt cttgctctac gtcctcctgc tggccttttg cgcctgtgca gtgggactga
      841 ttgccgtggg tgtcggggca cagcttgtcc tgagtcagac cataatccag ggggctaccc
      901 ctggctctct gttgccagtg gtcatcatcg cagtgggtgt cttcctcttc ctggtggctt
      961 ttgtgggctg ctgcggggcc tgcaaggaga actattgtct tatgatcacg tttgccatct
     1021 ttctgtctct tatcatgttg gtggaggtgg ccgcagccat tgctggctat gtgtttagag
     1081 ataaggtgat gtcagagttt aataacaact tccggcagca gatggagaat tacccgaaaa
     1141 acaaccacac tgcttcgatc ctggacagga tgcaggcaga ttttaagtgc tgtggggctg
     1201 ctaactacac agattgggag aaaatccctt ccatgtcgaa gaaccgagtc cccgactcct
     1261 gctgcattaa tgttactgtg ggctgtggga ttaatttcaa cgagaaggcg atccataagg
     1321 agggctgtgt ggagaagatt gggggctggc tgaggaaaaa tgtgctggtg gtagctgcag
     1381 cagcccttgg aattgctttt gtcgaggttt tgggaattgt ctttgcctgc tgcctcgtga
     1441 agagtatcag aagtggctac gaggtgatgg ctagcagtgg ctctggtagc atggcttcta
     1501 actttactca gttcgttctc gtcgacaatg gcggaactgg cgacgtgact gtcgccccaa
     1561 gcaacttcgc taacgggatc gctgaatgga tcagctctaa ctcgcgttca caggcttaca
     1621 aagtaacctg tagcgttcgt cagagctctg cgcagaatcg caaatacacc atcaaagtcg
     1681 aggtgcctaa aggcgcctgg cgttcgtact taaatatgga actaaccatt ccaattttcg
     1741 ccacgaattc cgactgcgag cttattgtta aggcaatgca aggtctccta aaagatggaa
     1801 acccgattcc ctcagcaatc gcagcgaata gcggtattta tgcaatggcg agcaatttca
     1861 cccagttcgt tctcgtcgac aatggcggaa ctggcgacgt gactgtcgcc ccaagcaact
     1921 tcgctaacgg gatcgctgaa tggatcagct ctaactcgcg ttcacaggct tacaaagtaa
     1981 cctgtagcgt tcgtcagagc tctgcgcaga atcgcaaata caccatcaaa gtcgaggtgc
     2041 ctaaaggcgc ctggcgttcg tacttaaata tggaactaac cattccaatt ttcgccacga
     2101 attccgactg cgagcttatt gttaaggcaa tgcaaggtct cctaaaagat ggaaacccga
     2161 ttccctcagc aatcgcagca aactccggca tctacggaag cggataccca tacgatgtgc
     2221 cagattacgc ttaaggatcg atccactagt ccagtgtggt ggaattctgc agatatccag
     2281 cacagtggcg gccgctcgag tctagagggc ccgtttaaac ccgctgatca gcctcgactg
     2341 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg
     2401 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga
     2461 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg
     2521 aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa
     2581 ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg
     2641 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     2701 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     2761 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     2821 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     2881 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     2941 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     3001 aaaatgagct gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt
     3061 agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     3121 ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     3181 catgcatctc aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct
     3241 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc
     3301 agaggccgag gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg
     3361 aggcctaggc ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca
     3421 gcacgtgatg aaaaagcctg aactcaccgc gacgtctgtc gagaagtttc tgatcgaaaa
     3481 gttcgacagc gtctccgacc tgatgcagct ctcggagggc gaagaatctc gtgctttcag
     3541 cttcgatgta ggagggcgtg gatatgtcct gcgggtaaat agctgcgccg atggtttcta
     3601 caaagatcgt tatgtttatc ggcactttgc atcggccgcg ctcccgattc cggaagtgct
     3661 tgacattggg gaattcagcg agagcctgac ctattgcatc tcccgccgtg cacagggtgt
     3721 cacgttgcaa gacctgcctg aaaccgaact gcccgctgtt ctgcagccgg tcgcggaggc
     3781 catggatgcg atcgctgcgg ccgatcttag ccagacgagc gggttcggcc cattcggacc
     3841 gcaaggaatc ggtcaataca ctacatggcg tgatttcata tgcgcgattg ctgatcccca
     3901 tgtgtatcac tggcaaactg tgatggacga caccgtcagt gcgtccgtcg cgcaggctct
     3961 cgatgagctg atgctttggg ccgaggactg ccccgaagtc cggcacctcg tgcacgcgga
     4021 tttcggctcc aacaatgtcc tgacggacaa tggccgcata acagcggtca ttgactggag
     4081 cgaggcgatg ttcggggatt cccaatacga ggtcgccaac atcttcttct ggaggccgtg
     4141 gttggcttgt atggagcagc agacgcgcta cttcgagcgg aggcatccgg agcttgcagg
     4201 atcgccgcgg ctccgggcgt atatgctccg cattggtctt gaccaactct atcagagctt
     4261 ggttgacggc aatttcgatg atgcagcttg ggcgcagggt cgatgcgacg caatcgtccg
     4321 atccggagcc gggactgtcg ggcgtacaca aatcgcccgc agaagcgcgg ccgtctggac
     4381 cgatggctgt gtagaagtac tcgccgatag tggaaaccga cgccccagca ctcgtccgag
     4441 ggcaaaggaa tagcacgtgc tacgagattt cgattccacc gccgccttct atgaaaggtt
     4501 gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat
     4561 gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt acaaataaag
     4621 caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt
     4681 gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta gctagagctt
     4741 ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca
     4801 caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact
     4861 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     4921 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
     4981 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     5041 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     5101 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     5161 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     5221 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     5281 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     5341 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     5401 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     5461 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     5521 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     5581 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     5641 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     5701 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     5761 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     5821 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     5881 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     5941 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     6001 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     6061 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     6121 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     6181 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     6241 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     6301 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     6361 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     6421 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     6481 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     6541 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     6601 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     6661 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     6721 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     6781 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     6841 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     6901 acctgacgtc gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc
     6961 tgctctgatg ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct
     7021 gagtagtgcg cgagcaaaat ttaagctaca acaag
//
