LOCUS       sequence-150321-        6305 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of EPN-01 assembly mutant L33R.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6305)
  AUTHORS   Votteler J, Ogohara C, Yi S, Hsia Y, Nattermann U, Belnap DM, King 
            NP, Sundquist WI
  TITLE     Designed proteins induce the formation of nanocage-containing 
            extracellular vesicles.
  JOURNAL   Nature. 2016 Dec 8;540(7632):292-295. doi: 10.1038/nature20607. Epub
            2016 Nov 30.
  PUBMED    27919066
REFERENCE   2  (bases 1 to 6305)
  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: 
            "10.1038/nature20607"; journalName: "Nature"; date: "2016-12-8- 8"; 
            volume: "540"; issue: "7632"; pages: "292-295"
COMMENT     Sequence Label: pCMV-EPN-01(Delta-I)
FEATURES             Location/Qualifiers
     source          1..6305
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1615..1644
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(1822..1841)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     primer_bind     complement(1896..1913)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1902..2126
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2172..2600
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2259..2278)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2469..2490
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2609..2629)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2614..2943
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2794..2929
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2856..2875
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3010..3804
                     /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(3064..3083)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3674..3693
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3978..4099
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4015..4034)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4069..4088
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4148..4164)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4148..4164)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4161..4183)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4172..4188
                     /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(4196..4202,4203..4220,4221..4226))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4241..4262
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4379..4396)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4550..5138)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4630..4649)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5309..6100,6101..6169))
                     /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     5932..5951
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6170..6274)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gtttaaacgg gccctctaga attatcgatc cggaggtacc cgggcggccg catgggtgcg
      961 cgtgcgtctg gttctaaatc tggctccggc tctgactctg gtagcaaaat cgaagagctg
     1021 ttcaagaaac acaagatcgt tgccgtgctg cgtgccaata gtgtggaaga agcgaaaaag
     1081 aaagcgctgg cggttttccg gggcggcgtt catctgattg aaattacctt taccgtgccg
     1141 gatgcggata ccgtgattaa ggaactgagc tttctgaagg aaatgggcgc gattattggt
     1201 gcgggcaccg tgaccagcgt ggagcagtgc cgtaaagcgg tggaaagtgg cgccgaattc
     1261 attgtgagtc cgcacctgga cgaggaaatt agccaatttt gcaaggagaa gggtgtgttc
     1321 tatatgccag gcgttatgac cccgaccgaa ctggtgaaag ccatgaaact gggccatacc
     1381 atcttaaaac tgtttccggg tgaggtggtg ggtccgcagt ttgttaaagc gatgaaaggt
     1441 ccgtttccga atgtgaaatt tgtgccaacc ggcggtgtta atctggacaa tgtgtgcgaa
     1501 tggttcaaag cgggcgtgct ggccgtgggc gtgggcagcg cgttagtgaa aggcaccccg
     1561 gtggaagtgg cggaaaaggc caaggcgttc gttgagaaga ttcgtggctg caccgaacag
     1621 aaactgatta gcgaagaaga tctgcagtct cgtccggaac cgaccgcacc gccggaagaa
     1681 tctttccgtt ctggtgttga aaccaccacg ccaccgcaaa aacaggagcc gatcgacaaa
     1741 gaactgtacc cgctgacctc tctgcgttct ctgttcggta acgacccgtc ttctcagtga
     1801 ctcgagaagc ttgctagcag atctttttcc ctctgccaaa aattatgggg acatcatgaa
     1861 gccccttgag cacttaagtt taaaccgctg atcagcctcg actgtgcctt ctagttgcca
     1921 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac
     1981 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat
     2041 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca
     2101 tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctctag
     2161 ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     2221 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     2281 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     2341 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     2401 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     2461 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     2521 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     2581 acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc
     2641 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg
     2701 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     2761 tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc
     2821 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc
     2881 tctgcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc
     2941 aaaaagctcc cgggagcttg tatatccatt ttcggatctg atcaagagac aggatgagga
     3001 tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag
     3061 aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc
     3121 cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg
     3181 aatgaactgc aggacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc
     3241 gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg
     3301 ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct
     3361 gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg
     3421 aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat
     3481 ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc
     3541 atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg
     3601 gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc
     3661 tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct
     3721 gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat
     3781 cgccttcttg acgagttctt ctgagcggga ctctggggtt cgaaatgacc gaccaagcga
     3841 cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct
     3901 tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg
     3961 agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     4021 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     4081 aactcatcaa tgtatcttat catgtctgta taccgtcgac ctctagctag agcttggcgt
     4141 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     4201 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
     4261 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     4321 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     4381 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     4441 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     4501 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     4561 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     4621 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     4681 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     4741 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     4801 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     4861 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     4921 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     4981 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     5041 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     5101 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     5161 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     5221 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     5281 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     5341 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     5401 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     5461 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     5521 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     5581 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     5641 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     5701 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     5761 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     5821 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     5881 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     5941 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     6001 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     6061 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     6121 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     6181 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     6241 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     6301 acgtc
//
