LOCUS       sequence-290881-        6717 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian Expression of 6xbsMS2-8xlet7.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6717)
  AUTHORS   Kuzuoglu-Ozturk D, Bhandari D, Huntzinger E, Fauser M, Helms S, 
            Izaurralde E
  TITLE     miRISC and the CCR4-NOT complex silence mRNA targets independently 
            of 43S ribosomal scanning.
  JOURNAL   EMBO J. 2016 Jun 1;35(11):1186-203. doi: 10.15252/embj.201592901. 
            Epub 2016 Mar 23.
  PUBMED    27009120
REFERENCE   2  (bases 1 to 6717)
  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: 
            "10.15252/embj.201592901"; journalName: "EMBO J"; date: "2016-06-1- 
            1"; volume: "35"; issue: "11"; pages: "1186-203"
COMMENT     Sequence Label: pCIneo-RLuc-6xbsMS2-8xlet7_AC
FEATURES             Location/Qualifiers
     source          1..6717
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        138..517
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        518..729
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     680..700
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     726..750
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          890..1022
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     1005..1024
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        1067..1085
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1089..1098
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1095..2030
                     /codon_start=1
                     /product="Renilla luciferase"
                     /label=hRluc
                     /note="human codon-optimized"
                     /translation="MASKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAEN
                     AVIFLHGNAASSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAW
                     FELLNLPKKIIFVGHDWGACLAFHYSYEHQDKIKAIVHAESVVDVIESWDEWPDIEEDI
                     ALIKSEEGEKMVLENNFFVETMLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPRE
                     IPLVKGGKPDVVQIVRNYNAYLRASDDLPKMFIESDPGFFSNAIVEGAKKFPNTEFVKV
                     KGLHFSQEDAPDEMGKYIKSFVERVLKNEQ"
     misc_RNA        2108..2126
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        2147..2165
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        2186..2204
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        2225..2243
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        2264..2282
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        2303..2321
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     rep_origin      2728..3183
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2815..2834)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3025..3046
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3300..3657
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3300..3491
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3323..3343)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3508..3643
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3708..4502
                     /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(3762..3781)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4372..4391
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4566..4614
                     /note="synthetic polyadenylation signal"
     primer_bind     complement(4655..4674)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4774..4796
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4834..4852)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4920..5024
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5025..5093,5094..5885)
                     /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     complement(5243..5262)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6056..6644
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6545..6564
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tcaatattgg ccattagcca tattattcat tggttatata gcataaatca atattggcta
       61 ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc
      121 aatatgaccg ccatgttggc attgattatt gactagttat taatagtaat caattacggg
      181 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
      241 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
      301 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
      361 ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga
      421 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg
      481 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac
      541 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
      601 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactg
      661 cgatcgcccg ccccgttgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata
      721 agcagagctc gtttagtgaa ccgtcagatc actagaagct ttattgcggt agtttatcac
      781 agttaaattg ctaacgcagt cagtgcttct gacacaacag tctcgaactt aagctgcagt
      841 gactctctta aggtagcctt gcagaagttg gtcgtgaggc actgggcagg taagtatcaa
      901 ggttacaaga caggtttaag gagaccaata gaaactgggc ttgtcgagac agagaagact
      961 cttgcgtttc tgataggcac ctattggtct tactgacatc cactttgcct ttctctccac
     1021 aggtgtccac tcccagttca attacagctc ttaaggctag agtacttaat acgactcact
     1081 ataggctagc caccatggct tccaaggtgt acgaccccga gcaacgcaaa cgcatgatca
     1141 ctgggcctca gtggtgggct cgctgcaagc aaatgaacgt gctggactcc ttcatcaact
     1201 actatgattc cgagaagcac gccgagaacg ccgtgatttt tctgcatggt aacgctgcct
     1261 ccagctacct gtggaggcac gtcgtgcctc acatcgagcc cgtggctaga tgcatcatcc
     1321 ctgatctgat cggaatgggt aagtccggca agagcgggaa tggctcatat cgcctcctgg
     1381 atcactacaa gtacctcacc gcttggttcg agctgctgaa ccttccaaag aaaatcatct
     1441 ttgtgggcca cgactggggg gcttgtctgg cctttcacta ctcctacgag caccaagaca
     1501 agatcaaggc catcgtccat gctgagagtg tcgtggacgt gatcgagtcc tgggacgagt
     1561 ggcctgacat cgaggaggat atcgccctga tcaagagcga agagggcgag aaaatggtgc
     1621 ttgagaataa cttcttcgtc gagaccatgc tcccaagcaa gatcatgcgg aaactggagc
     1681 ctgaggagtt cgctgcctac ctggagccat tcaaggagaa gggcgaggtt agacggccta
     1741 ccctctcctg gcctcgcgag atccctctcg ttaagggagg caagcccgac gtcgtccaga
     1801 ttgtccgcaa ctacaacgcc taccttcggg ccagcgacga tctgcctaag atgttcatcg
     1861 agtccgaccc tgggttcttt tccaacgcta ttgtcgaggg agctaagaag ttccctaaca
     1921 ccgagttcgt gaaggtgaag ggcctccact tcagccagga ggacgctcca gatgaaatgg
     1981 gtaagtacat caagagcttc gtggagcgcg tgctgaagaa cgagcagtaa ttctagccga
     2041 ctcactatag ggcgaattgg cggaaggccg tcaaggccgc atcaacgagc tctctagact
     2101 gtagaaaaca tgaggatcac ccatgtctgc tggacgactg tagaaaacat gaggatcacc
     2161 catgtctgct gtctagctgt agaaaacatg aggatcaccc atgtctgctg gacgactgta
     2221 gaaaacatga ggatcaccca tgtctgctgt ctagctgtag aaaacatgag gatcacccat
     2281 gtctgctgga cgactgtaga aaacatgagg atcacccatg tctgctgggc ccgcttcgtg
     2341 gttctggcga tcgatcgaga ctatacaagg atctacctca gtcgagacta tacaaggatc
     2401 tacctcagtc gagactatac aaggatctac ctcagtcgag actatacaag gatctacctc
     2461 agtcgagact atacaaggat ctacctcagt cgagactata caaggatcta cctcagtcga
     2521 gactatacaa ggatctacct cagtcgagac tatacaagga tctacctcag tcgacgcggc
     2581 cgctggccgc ctcgaggcgg ccgcttccct ttagtgaggg ttaatgcttc gagaaatccg
     2641 ataaggatcg atccgggctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa
     2701 cagttgcgca gcctgaatgg cgaatggacg cgccctgtag cggcgcatta agcgcggcgg
     2761 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     2821 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     2881 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     2941 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     3001 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     3061 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     3121 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa
     3181 tttcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatacgcgg
     3241 atctgcgcag caccatggcc tgaaataacc tctgaaagag gaacttggtt aggtaccttc
     3301 tgaggcggaa agaaccagct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc
     3361 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga
     3421 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     3481 accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat
     3541 tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc
     3601 tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag
     3661 cttgattctt ctgacacaac agtctcgaac ttaaggctag agccaccatg attgaacaag
     3721 atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg
     3781 cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc
     3841 cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag
     3901 cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca
     3961 ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat
     4021 ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata
     4081 cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac
     4141 gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc
     4201 tcgcgccagc cgaactgttc gccaggctca aggcgcgcat gcccgacggc gaggatctcg
     4261 tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg
     4321 gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta
     4381 cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg
     4441 gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct
     4501 gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgatg
     4561 gccgcaataa aatatcttta ttttcattac atctgtgtgt tggttttttg tgtgaatcga
     4621 tagcgataag gatccgcgta tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     4681 taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
     4741 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
     4801 caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg
     4861 ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc
     4921 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     4981 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     5041 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     5101 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     5161 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     5221 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
     5281 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     5341 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     5401 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     5461 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     5521 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
     5581 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     5641 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     5701 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     5761 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     5821 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     5881 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     5941 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     6001 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     6061 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     6121 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     6181 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     6241 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     6301 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     6361 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     6421 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     6481 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     6541 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     6601 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     6661 cctttttacg gttcctggcc ttttgctggc cttttgctca catggctcga cagatct
//
