LOCUS       sequence-357072-        6191 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of EGFP(W58stop)-HiBiT.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6191)
  AUTHORS   Somiya M, Kuroda S
  TITLE     Verification of extracellular vesicle-mediated functional mRNA 
            delivery by RNA editing
  JOURNAL   bioRxiv 2022.01.25.477620
REFERENCE   2  (bases 1 to 6191)
  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: "bioRxiv 
            2022.01.25.477620"
COMMENT     Sequence Label: pcDNA3.1-EGFP(W58stop)-HiBiT
FEATURES             Location/Qualifiers
     source          1..6191
                     /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"
     regulatory      986..995
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(992..994,995..997,998..1708)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVP*PTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1037..1058)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1298..1317)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1645..1666
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1782..1799)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1788..2012
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2058..2486
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2145..2164)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2355..2376
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2495..2515)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2500..2829
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2680..2815
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2742..2761
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2896..3690
                     /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(2950..2969)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3560..3579
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3864..3985
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3901..3920)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3955..3974
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4034..4050)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4034..4050)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4047..4069)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4058..4074
                     /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(4082..4088,4089..4106,4107..4112))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4127..4148
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4265..4282)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4436..5024)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4516..4535)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5195..5986,5987..6055))
                     /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     5818..5837
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6056..6160)
                     /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 gtttaaactt aagcttggta ccgagctcgg atccactagt ccagtgtggt ggaattctgc
      961 agatatccag cacagtggcg gccgcgccac catggtgagc aagggcgagg agctgttcac
     1021 cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt
     1081 gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac
     1141 caccggcaag ctgcccgtgc cctagcccac cctcgtgacc accctgacct acggcgtgca
     1201 gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc
     1261 cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg
     1321 cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga
     1381 cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa
     1441 cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca
     1501 caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg
     1561 cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa
     1621 agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat
     1681 cactctcggc atggacgagc tgtacaaggt gagcggctgg cggctgttca agaagattag
     1741 ctgactcgag tctagagggc ccgtttaaac ccgctgatca gcctcgactg tgccttctag
     1801 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac
     1861 tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca
     1921 ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag
     1981 caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg
     2041 ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     2101 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     2161 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     2221 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     2281 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     2341 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     2401 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     2461 gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga
     2521 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     2581 accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     2641 aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc
     2701 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
     2761 gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc
     2821 ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca agagacagga
     2881 tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg
     2941 gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc
     3001 gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt
     3061 gccctgaatg aactgcagga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt
     3121 ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc
     3181 gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc
     3241 atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac
     3301 caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag
     3361 gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag
     3421 gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat
     3481 atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg
     3541 gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa
     3601 tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc
     3661 ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa atgaccgacc
     3721 aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt
     3781 tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca
     3841 tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa
     3901 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     3961 tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct
     4021 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     4081 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     4141 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     4201 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     4261 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     4321 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     4381 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     4441 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     4501 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     4561 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     4621 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     4681 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     4741 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     4801 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     4861 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     4921 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     4981 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     5041 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     5101 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     5161 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     5221 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     5281 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     5341 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     5401 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     5461 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     5521 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     5581 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     5641 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     5701 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     5761 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     5821 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     5881 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     5941 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     6001 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     6061 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     6121 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     6181 cacctgacgt c
//
