LOCUS       sequence-350186-        7559 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of heat shock 70 kDa protein 8 fused to a 
            C-terminal HA tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7559)
  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 7559)
  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: pMEH365 Hspa8 HA
FEATURES             Location/Qualifiers
     source          1..7559
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     551..570
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(789..893)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(968..987)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1159..1538
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1539..1742
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1693..1713
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1787..1806
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1787..1805
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             3778..3804
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(3905..3922)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3911..4135
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4181..4609
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4268..4287)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4478..4499
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4618..4638)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4623..4952
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4803..4938
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4865..4884
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             5001..6026
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             5001..6026
                     /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    6156..6277
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6193..6212)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6247..6266
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6326..6342)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6326..6342)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6339..6361)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6350..6366
                     /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(6374..6380,6381..6398,6399..6404))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6419..6440
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6557..6574)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6728..7316)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6808..6827)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7487..7559,1..719,720..788))
                     /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"
ORIGIN
        1 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
       61 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
      121 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
      181 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
      241 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
      301 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
      361 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
      421 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
      481 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
      541 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
      601 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
      661 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
      721 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
      781 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
      841 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
      901 ccccgaaaag tgccacctga cgtcgacgga tcgggagatc tcccgatccc ctatggtgca
      961 ctctcagtac aatctgctct gatgccgcat agttaagcca gtatctgctc cctgcttgtg
     1021 tgttggaggt cgctgagtag tgcgcgagca aaatttaagc tacaacaagg caaggcttga
     1081 ccgacaattg catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac
     1141 gggccagata tacgcgttga cattgattat tgactagtta ttaatagtaa tcaattacgg
     1201 ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc
     1261 cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca
     1321 tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg
     1381 cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg
     1441 acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt
     1501 ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca
     1561 tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg
     1621 tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact
     1681 ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag
     1741 ctctctggct aactagagaa cccactgctt actggcttat cgaaattaat acgactcact
     1801 atagggagac ccaagctggg taccatgtcc aagggacctg cagttggtat tgatcttggc
     1861 accacctact cttgtgtggg tgttttccag cacggaaaag tcgagataat tgccaatgat
     1921 cagggaaacc gaaccactcc aagctatgtc gcctttacgg acactgaacg gttgatcggt
     1981 gatgccgcaa agaatcaagt tgcaatgaac cccaccaaca cagtttttga tgccaaacgt
     2041 ctgattggac gcagatttga tgatgctgtt gtccagtctg atatgaaaca ttggcccttt
     2101 atggtggtga atgatgctgg caggcccaag gtccaagtag aatacaaggg agagaccaaa
     2161 agcttctatc cagaggaggt gtcttctatg gttctgacaa agatgaagga aattgcagaa
     2221 gcctaccttg ggaagactgt taccaatgct gtggtcacag tgccagctta ctttaatgac
     2281 tctcagcgtc aggctaccaa agatgctgga actattgctg gtctcaatgt acttagaatt
     2341 attaatgagc caactgctgc tgctattgct tacggcttag acaaaaaggt tggagcagaa
     2401 agaaacgtgc tcatctttga cctgggaggt ggcacttttg atgtgtcaat cctcactatt
     2461 gaggatggaa tctttgaggt caagtctaca gctggagaca cccacttggg tggagaagat
     2521 tttgacaacc gaatggtcaa ccattttatt gctgagttta agcgcaagca taagaaggac
     2581 atcagtgaga acaagagagc tgtaagacgc ctccgtactg cttgtgaacg tgctaagcgt
     2641 accctctctt ccagcaccca ggccagtatt gagatcgatt ctctctatga aggaatcgac
     2701 ttctatacct ccattacccg tgcccgattt gaagaactga atgctgacct gttccgtggc
     2761 accctggacc cagtagagaa agcccttcga gatgccaaac tagacaagtc acagattcat
     2821 gatattgtcc tggttggtgg ttctactcgt atccccaaga ttcagaagct tctccaagac
     2881 ttcttcaatg gaaaagaact gaataagagc atcaaccctg atgaagctgt tgcttatggt
     2941 gcagctgtcc aggcagccat cttgtctgga gacaagtctg agaatgttca agatttgctg
     3001 ctcttggatg tcactcctct ttcccttggt attgaaactg ctggtggagt catgactgtc
     3061 ctcatcaagc gtaataccac cattcctacc aagcagacac agaccttcac tacctattct
     3121 gacaaccagc ctggtgtgct tattcaggtt tatgaaggcg agcgtgccat gacaaaggat
     3181 aacaacctgc ttggcaagtt tgaactcaca ggcatacctc ctgcaccccg aggtgttcct
     3241 cagattgaag tcacttttga cattgatgcc aatggtatac tcaatgtctc tgctgtggac
     3301 aagagtacgg gaaaagagaa caagattact atcactaatg acaagggccg tttgagcaag
     3361 gaagacattg aacgtatggt ccaggaagct gagaagtaca aagctgaaga tgagaagcag
     3421 agggacaagg tgtcatccaa gaattcactt gagtcctatg ccttcaacat gaaagcaact
     3481 gttgaagatg agaaacttca aggcaagatt aacgatgagg acaaacagaa gattctggac
     3541 aagtgtaatg aaattatcaa ctggcttgat aagaatcaga ctgctgagaa ggaagaattt
     3601 gaacatcaac agaaagagct ggagaaagtt tgcaacccca tcatcaccaa gctgtaccag
     3661 agtgcaggag gcatgccagg aggaatgcct gggggatttc ctggtggtgg agctcctccc
     3721 tctggtggtg cttcctcagg gcccaccatt gaagaggttg atgctagcgg aagcggatac
     3781 ccatacgatg tgccagatta cgcttaagga tcgatccact agtccagtgt ggtggaattc
     3841 tgcagatatc cagcacagtg gcggccgctc gagtctagag ggcccgttta aacccgctga
     3901 tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct
     3961 tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca
     4021 tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag
     4081 ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct
     4141 gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca
     4201 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     4261 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     4321 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     4381 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     4441 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     4501 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
     4561 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaatta attctgtgga
     4621 atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
     4681 gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca
     4741 gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc
     4801 ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt
     4861 tttttattta tgcagaggcc gaggccgcct ctgcctctga gctattccag aagtagtgag
     4921 gaggcttttt tggaggccta ggcttttgca aaaagctccc gggagcttgt atatccattt
     4981 tcggatctga tcagcacgtg atgaaaaagc ctgaactcac cgcgacgtct gtcgagaagt
     5041 ttctgatcga aaagttcgac agcgtctccg acctgatgca gctctcggag ggcgaagaat
     5101 ctcgtgcttt cagcttcgat gtaggagggc gtggatatgt cctgcgggta aatagctgcg
     5161 ccgatggttt ctacaaagat cgttatgttt atcggcactt tgcatcggcc gcgctcccga
     5221 ttccggaagt gcttgacatt ggggaattca gcgagagcct gacctattgc atctcccgcc
     5281 gtgcacaggg tgtcacgttg caagacctgc ctgaaaccga actgcccgct gttctgcagc
     5341 cggtcgcgga ggccatggat gcgatcgctg cggccgatct tagccagacg agcgggttcg
     5401 gcccattcgg accgcaagga atcggtcaat acactacatg gcgtgatttc atatgcgcga
     5461 ttgctgatcc ccatgtgtat cactggcaaa ctgtgatgga cgacaccgtc agtgcgtccg
     5521 tcgcgcaggc tctcgatgag ctgatgcttt gggccgagga ctgccccgaa gtccggcacc
     5581 tcgtgcacgc ggatttcggc tccaacaatg tcctgacgga caatggccgc ataacagcgg
     5641 tcattgactg gagcgaggcg atgttcgggg attcccaata cgaggtcgcc aacatcttct
     5701 tctggaggcc gtggttggct tgtatggagc agcagacgcg ctacttcgag cggaggcatc
     5761 cggagcttgc aggatcgccg cggctccggg cgtatatgct ccgcattggt cttgaccaac
     5821 tctatcagag cttggttgac ggcaatttcg atgatgcagc ttgggcgcag ggtcgatgcg
     5881 acgcaatcgt ccgatccgga gccgggactg tcgggcgtac acaaatcgcc cgcagaagcg
     5941 cggccgtctg gaccgatggc tgtgtagaag tactcgccga tagtggaaac cgacgcccca
     6001 gcactcgtcc gagggcaaag gaatagcacg tgctacgaga tttcgattcc accgccgcct
     6061 tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc
     6121 gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg
     6181 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     6241 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct
     6301 ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     6361 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     6421 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     6481 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     6541 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     6601 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     6661 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     6721 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     6781 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     6841 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     6901 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     6961 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     7021 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     7081 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     7141 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     7201 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     7261 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     7321 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     7381 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     7441 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     7501 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactc
//
