LOCUS       sequence-357071-        5906 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Mammalian expression of Nluc(W12stop).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5906)
  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 5906)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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-Nluc(W12stop)
FEATURES             Location/Qualifiers
     source          1..5906
                     /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      935..944
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             941..1453
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGD*RQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     CDS             1454..1477
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(1497..1514)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1503..1727
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1773..2201
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1860..1879)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2070..2091
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2210..2230)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2215..2544
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2395..2530
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2457..2476
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2611..3405
                     /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
                     LATRDIAEELGGEWADRFLVLYGIAAPYSQRIAFYRLLDEFF"
     primer_bind     complement(2665..2684)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3275..3294
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3579..3700
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3616..3635)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3670..3689
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3749..3765)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3749..3765)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3762..3784)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3773..3789
                     /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(3797..3803,3804..3821,3822..3827))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3842..3863
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3980..3997)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4151..4739)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4231..4250)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4910..5701,5702..5770))
                     /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     5533..5552
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5771..5875)
                     /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 atccgccacc atggtcttca cactcgaaga
      961 tttcgttggg gactagcgac agacagccgg ctacaacctg gaccaagtcc ttgaacaggg
     1021 aggtgtgtcc agtttgtttc agaatctcgg ggtgtccgta actccgatcc aaaggattgt
     1081 cctgagcggt gaaaatgggc tgaagatcga catccatgtc atcatcccgt atgaaggtct
     1141 gagcggcgac caaatgggcc agatcgaaaa aatttttaag gtggtgtacc ctgtggatga
     1201 tcatcacttt aaggtgatcc tgcactatgg cacactggta atcgacgggg ttacgccgaa
     1261 catgatcgac tatttcggac ggccgtatga aggcatcgcc gtgttcgacg gcaaaaagat
     1321 cactgtaaca gggaccctgt ggaacggcaa caaaattatc gacgagcgcc tgatcaaccc
     1381 cgacggctcc ctgctgttcc gagtaaccat caacggagtg accggctggc ggctgtgtga
     1441 acgcattctg gcggattaca aggatgacga cgataagtga taaacccgct gatcagcctc
     1501 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
     1561 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg
     1621 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga
     1681 ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga
     1741 aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc
     1801 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     1861 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     1921 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     1981 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     2041 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     2101 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     2161 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt
     2221 cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     2281 ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     2341 caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg
     2401 cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt
     2461 tatgcagagg ccgaggccgc ctctgcctct gagctattcc agaagtagtg aggaggcttt
     2521 tttggaggcc taggcttttg caaaaagctc ccgggagctt gtatatccat tttcggatct
     2581 gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt
     2641 tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc
     2701 tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag
     2761 accgacctgt ccggtgccct gaatgaactg caggacgagg cagcgcggct atcgtggctg
     2821 gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac
     2881 tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc
     2941 gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc
     3001 tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc
     3061 ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg
     3121 ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat
     3181 gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc
     3241 cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa
     3301 gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctccctat
     3361 tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt
     3421 tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat tccaccgccg
     3481 ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc
     3541 agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata
     3601 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     3661 attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga
     3721 cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     3781 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     3841 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     3901 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     3961 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     4021 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     4081 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     4141 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     4201 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     4261 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     4321 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     4381 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     4441 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     4501 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     4561 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     4621 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     4681 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     4741 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     4801 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     4861 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     4921 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     4981 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     5041 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     5101 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     5161 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     5221 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     5281 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     5341 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     5401 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     5461 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     5521 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     5581 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     5641 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     5701 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     5761 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     5821 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     5881 ttccccgaaa agtgccacct gacgtc
//
