LOCUS       sequence-431663-        6979 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of FLAG/HA tagged firefly luciferase (FL).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6979)
  AUTHORS   Nishi K, Takahashi T, Suzawa M, Miyakawa T, Nagasawa T, Ming Y, 
            Tanokura M, Ui-Tei K
  TITLE     Control of the localization and function of a miRNA silencing 
            component TNRC6A by Argonaute protein.
  JOURNAL   Nucleic Acids Res. 2015 Nov 16;43(20):9856-73. doi: 
            10.1093/nar/gkv1026. Epub 2015 Oct 7.
  PUBMED    26446993
REFERENCE   2  (bases 1 to 6979)
  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: 
            "10.1093/nar/gkv1026"; journalName: "Nucleic Acids Res"; date: 
            "2015-11-16- 16"; volume: "43"; issue: "20"; pages: "9856-73"
COMMENT     Sequence Label: pIRESneo-FLAG/HA-FL
FEATURES             Location/Qualifiers
     source          1..6979
                     /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      922..931
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             931..954
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             955..981
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             991..2643
                     /codon_start=1
                     /gene="luc+"
                     /product="firefly luciferase"
                     /label=luciferase
                     /note="enhanced luc+ version of the luciferase gene"
                     /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA
                     HIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP
                     ANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS
                     MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
                     RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK
                     IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY
                     GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS
                     GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL
                     LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV
                     FVDEVPKGLTGKLDARKIREILIKAKKGGKIAV"
     primer_bind     complement(1057..1075)
                     /label=LucNrev
                     /note="Firefly luciferase, reverse primer"
     intron          2720..2949
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     primer_bind     2932..2951
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    join(3022..3596,3597..3599,3600..3608)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3189..3206)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3416..3435
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3631..4434
                     /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="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3694..3713)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4304..4323
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4466..4483)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4472..4696
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     4743..4762
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4822..4838)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4822..4838)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4835..4857)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4846..4862
                     /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(4870..4876,4877..4894,4895..4900))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4915..4936
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5053..5070)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5224..5812)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5304..5323)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5983..6774,6775..6843))
                     /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     6606..6625
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6844..6948)
                     /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 gcttggtacc
      901 gagctcggat cgatatcgcc ggccgccatg gactacaagg acgacgatga caagtaccct
      961 tatgacgtgc ccgattacgc tagcggccgc atggaagacg ccaaaaacat aaagaaaggc
     1021 ccggcgccat tctatccgct ggaagatgga accgctggag agcaactgca taaggctatg
     1081 aagagatacg ccctggttcc tggaacaatt gcttttacag atgcacatat cgaggtggac
     1141 atcacttacg ctgagtactt cgaaatgtcc gttcggttgg cagaagctat gaaacgatat
     1201 gggctgaata caaatcacag aatcgtcgta tgcagtgaaa actctcttca attctttatg
     1261 ccggtgttgg gcgcgttatt tatcggagtt gcagttgcgc ccgcgaacga catttataat
     1321 gaacgtgaat tgctcaacag tatgggcatt tcgcagccta ccgtggtgtt cgtttccaaa
     1381 aaggggttgc aaaaaatttt gaacgtgcaa aaaaagctcc caatcatcca aaaaattatt
     1441 atcatggatt ctaaaacgga ttaccaggga tttcagtcga tgtacacgtt cgtcacatct
     1501 catctacctc ccggttttaa tgaatacgat tttgtgccag agtccttcga tagggacaag
     1561 acaattgcac tgatcatgaa ctcctctgga tctactggtc tgcctaaagg tgtcgctctg
     1621 cctcatagaa ctgcctgcgt gagattctcg catgccagag atcctatttt tggcaatcaa
     1681 atcattccgg atactgcgat tttaagtgtt gttccattcc atcacggttt tggaatgttt
     1741 actacactcg gatatttgat atgtggattt cgagtcgtct taatgtatag atttgaagaa
     1801 gagctgtttc tgaggagcct tcaggattac aagattcaaa gtgcgctgct ggtgccaacc
     1861 ctattctcct tcttcgccaa aagcactctg attgacaaat acgatttatc taatttacac
     1921 gaaattgctt ctggtggcgc tcccctctct aaggaagtcg gggaagcggt tgccaagagg
     1981 ttccatctgc caggtatcag gcaaggatat gggctcactg agactacatc agctattctg
     2041 attacacccg agggggatga taaaccgggc gcggtcggta aagttgttcc attttttgaa
     2101 gcgaaggttg tggatctgga taccgggaaa acgctgggcg ttaatcaaag aggcgaactg
     2161 tgtgtgagag gtcctatgat tatgtccggt tatgtaaaca atccggaagc gaccaacgcc
     2221 ttgattgaca aggatggatg gctacattct ggagacatag cttactggga cgaagacgaa
     2281 cacttcttca tcgttgaccg cctgaagtct ctgattaagt acaaaggcta tcaggtggct
     2341 cccgctgaat tggaatccat cttgctccaa caccccaaca tcttcgacgc aggtgtcgca
     2401 ggtcttcccg acgatgacgc cggtgaactt cccgccgccg ttgttgtttt ggagcacgga
     2461 aagacgatga cggaaaaaga gatcgtggat tacgtcgcca gtcaagtaac aaccgcgaaa
     2521 aagttgcgcg gaggagttgt gtttgtggac gaagtaccga aaggtcttac cggaaaactc
     2581 gacgcaagaa aaatcagaga gatcctcata aaggccaaga agggcggaaa gatcgccgtg
     2641 taagaattca gtggatccac tagtaacggc cgccagtgtg ctggaattaa ttcgctgtct
     2701 gcgagggcca gctgttgggg tgagtactcc ctctcaaaag cgggcatgac ttctgcgcta
     2761 agattgtcag tttccaaaaa cgaggaggat ttgatattca cctggcccgc ggtgatgcct
     2821 ttgagggtgg ccgcgtccat ctggtcagaa aagacaatct ttttgttgtc aagcttgagg
     2881 tgtggcaggc ttgagatctg gccatacact tgagtgacaa tgacatccac tttgcctttc
     2941 tctccacagg tgtccactcc caggtccaac tgcaggtcga gcatgcatct agggcggcca
     3001 attccgcccc tctccccccc ccccctctcc ctcccccccc cctaacgtta ctggccgaag
     3061 ccgcttggaa taaggccggt gtgcgtttgt ctatatgtta ttttccacca tattgccgtc
     3121 ttttggcaat gtgagggccc ggaaacctgg ccctgtcttc ttgacgagca ttcctagggg
     3181 tctttcccct ctcgccaaag gaatgcaagg tctgttgaat gtcgtgaagg aagcagttcc
     3241 tctggaagct tcttgaagac aaacaacgtc tgtagcgacc ctttgcaggc agcggaaccc
     3301 cccacctggc gacaggtgcc tctgcggcca aaagccacgt gtataagata cacctgcaaa
     3361 ggcggcacaa ccccagtgcc acgttgtgag ttggatagtt gtggaaagag tcaaatggct
     3421 ctcctcaagc gtattcaaca aggggctgaa ggatgcccag aaggtacccc attgtatggg
     3481 atctgatctg gggcctcggt gcacatgctt tacatgtgtt tagtcgaggt taaaaaaacg
     3541 tctaggcccc ccgaaccacg gggacgtggt tttcctttga aaaacacgat gataagcttg
     3601 ccacaacccg ggataattcc tgcagccaat atgggatcgg ccattgaaca agatggattg
     3661 cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag
     3721 acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt
     3781 tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta
     3841 tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg
     3901 ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt
     3961 gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat
     4021 ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg
     4081 atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca
     4141 gccgaactgt tcgccaggct caaggcgcgc atgcccgacg gcgatgatct cgtcgtgacc
     4201 catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc
     4261 gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat
     4321 attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc
     4381 gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgaggggat
     4441 caattctcta gagctcgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt
     4501 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc
     4561 taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt
     4621 ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat
     4681 gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctcgag tgcattctag
     4741 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag
     4801 ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     4861 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     4921 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     4981 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     5041 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     5101 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     5161 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     5221 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     5281 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     5341 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     5401 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     5461 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     5521 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     5581 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     5641 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     5701 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     5761 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     5821 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     5881 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     5941 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     6001 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     6061 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     6121 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     6181 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     6241 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     6301 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     6361 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     6421 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     6481 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     6541 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     6601 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     6661 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     6721 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     6781 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     6841 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     6901 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     6961 aaaagtgcca cctgacgtc
//
