LOCUS       sequence-319293-        6308 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  EPN-01 tagged with HiBiT tag under CMV promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6308)
  AUTHORS   Somiya M, Kuroda S
  TITLE     Real-Time Luminescence Assay for Cytoplasmic Cargo Delivery of 
            Extracellular Vesicles.
  JOURNAL   Anal Chem. 2021 Mar 24. doi: 10.1021/acs.analchem.1c00339.
  PUBMED    33759512
REFERENCE   2  (bases 1 to 6308)
  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: "Anal Chem. 
            2021 Mar 24. doi: 10.1021/acs.analchem.1c00339."
COMMENT     Sequence Label: pCMV-EPN-01(HiBiT)
FEATURES             Location/Qualifiers
     source          1..6308
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1591..1610)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     primer_bind     complement(1665..1682)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1671..1895
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1941..2369
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2028..2047)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2238..2259
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2378..2398)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2383..2712
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2563..2698
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2625..2644
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2779..3573
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2833..2852)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3443..3462
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3747..3868
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3784..3803)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3838..3857
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3917..3933)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3917..3933)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3930..3952)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3941..3957
                     /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(3965..3971,3972..3989,3990..3995))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4010..4031
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4148..4165)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4319..4907)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4399..4418)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5078..5869,5870..5938))
                     /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     5701..5720
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5939..6043)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6118..6137)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctg
      661 gctagcgttt aaacgggccc tctagaatta tcgatccgga ggtacccggg cggccgcatg
      721 ggtgcgcgtg cgtctggttc taaatctggc tccggctctg actctggtag caaaatcgaa
      781 gagctgttca agaaacacaa gatcgttgcc gtgctgcgtg ccaatagtgt ggaagaagcg
      841 aaaaagaaag cgctggcggt tttcctgggc ggcgttcatc tgattgaaat tacctttacc
      901 gtgccggatg cggataccgt gattaaggaa ctgagctttc tgaaggaaat gggcgcgatt
      961 attggtgcgg gcaccgtgac cagcgtggag cagtgccgta aagcggtgga aagtggcgcc
     1021 gaattcattg tgagtccgca cctggacgag gaaattagcc aattttgcaa ggagaagggt
     1081 gtgttctata tgccaggcgt tatgaccccg accgaactgg tgaaagccat gaaactgggc
     1141 cataccatct taaaactgtt tccgggtgag gtggtgggtc cgcagtttgt taaagcgatg
     1201 aaaggtccgt ttccgaatgt gaaatttgtg ccaaccggcg gtgttaatct ggacaatgtg
     1261 tgcgaatggt tcaaagcggg cgtgctggcc gtgggcgtgg gcagcgcgtt agtgaaaggc
     1321 accccggtgg aagtggcgga aaaggccaag gcgttcgttg agaagattcg tggctgcacc
     1381 gtgagcggct ggcggctgtt caagaagatt agccagtctc gtccggaacc gaccgcaccg
     1441 ccggaagaat ctttccgttc tggtgttgaa accaccacgc caccgcaaaa acaggagccg
     1501 atcgacaaag aactgtaccc gctgacctct ctgcgttctc tgttcggtaa cgacccgtct
     1561 tctcagtgac tcgagaagct tgctagcaga tctttttccc tctgccaaaa attatgggga
     1621 catcatgaag ccccttgagc acttaagttt aaaccgctga tcagcctcga ctgtgccttc
     1681 tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc
     1741 cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg
     1801 tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa
     1861 tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg
     1921 gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     1981 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
     2041 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     2101 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg
     2161 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     2221 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
     2281 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
     2341 gctgatttaa caaaaattta acgcgaatta attctgtgga atgtgtgtca gttagggtgt
     2401 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     2461 gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     2521 ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg
     2581 cccagttccg cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc
     2641 gaggccgcct ctgcctctga gctattccag aagtagtgag gaggcttttt tggaggccta
     2701 ggcttttgca aaaagctccc gggagcttgt atatccattt tcggatctga tcaagagaca
     2761 ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct
     2821 tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc
     2881 gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc
     2941 ggtgccctga atgaactgca ggacgaggca gcgcggctat cgtggctggc cacgacgggc
     3001 gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg
     3061 ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc
     3121 atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac
     3181 caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat
     3241 caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc
     3301 aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg
     3361 aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg
     3421 gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc
     3481 gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc
     3541 gccttctatc gccttcttga cgagttcttc tgagcgggac tctggggttc gaaatgaccg
     3601 accaagcgac gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa
     3661 ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc
     3721 tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat
     3781 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     3841 gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga
     3901 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     3961 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     4021 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     4081 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     4141 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     4201 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     4261 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     4321 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     4381 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     4441 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     4501 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     4561 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     4621 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     4681 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     4741 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     4801 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     4861 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     4921 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     4981 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     5041 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     5101 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     5161 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     5221 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     5281 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     5341 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     5401 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     5461 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     5521 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     5581 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     5641 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     5701 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     5761 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     5821 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     5881 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     5941 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     6001 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     6061 tgccacctga cgtcgacgga tcgggagatc tcccgatccc ctatggtgca ctctcagtac
     6121 aatctgctct gatgccgcat agttaagcca gtatctgctc cctgcttgtg tgttggaggt
     6181 cgctgagtag tgcgcgagca aaatttaagc tacaacaagg caaggcttga ccgacaattg
     6241 catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac gggccagata
     6301 tacgcgtt
//
