LOCUS       sequence-357753-        6939 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian Expression of 6xbsMS2-random.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6939)
  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 6939)
  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-random_AC
FEATURES             Location/Qualifiers
     source          1..6939
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        77..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..592
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     543..563
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     589..613
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          753..885
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     868..887
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        930..948
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      952..961
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             958..1893
                     /codon_start=1
                     /product="Renilla luciferase"
                     /label=hRluc
                     /note="human codon-optimized"
                     /translation="MASKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAEN
                     AVIFLHGNAASSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAW
                     FELLNLPKKIIFVGHDWGACLAFHYSYEHQDKIKAIVHAESVVDVIESWDEWPDIEEDI
                     ALIKSEEGEKMVLENNFFVETMLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPRE
                     IPLVKGGKPDVVQIVRNYNAYLRASDDLPKMFIESDPGFFSNAIVEGAKKFPNTEFVKV
                     KGLHFSQEDAPDEMGKYIKSFVERVLKNEQ"
     misc_RNA        1971..1989
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        2010..2028
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        2049..2067
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        2088..2106
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        2127..2145
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        2166..2184
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     polyA_signal    2506..2627
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2517..2536)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2571..2590
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      2813..3268
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2900..2919)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3110..3131
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3385..3742
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3385..3576
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3408..3428)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3593..3728
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3793..4587
                     /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(3847..3866)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4457..4476
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4651..4699
                     /note="synthetic polyadenylation signal"
     primer_bind     complement(4740..4759)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4859..4881
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4919..4937)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5005..5109
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5110..5178,5179..5970)
                     /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(5328..5347)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6141..6729
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6630..6649
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ggcattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt ccgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttacggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta caccaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctgcgatcgc ccgccccgtt
      541 gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctcgtttagt
      601 gaaccgtcag atcactagaa gctttattgc ggtagtttat cacagttaaa ttgctaacgc
      661 agtcagtgct tctgacacaa cagtctcgaa cttaagctgc agtgactctc ttaaggtagc
      721 cttgcagaag ttggtcgtga ggcactgggc aggtaagtat caaggttaca agacaggttt
      781 aaggagacca atagaaactg ggcttgtcga gacagagaag actcttgcgt ttctgatagg
      841 cacctattgg tcttactgac atccactttg cctttctctc cacaggtgtc cactcccagt
      901 tcaattacag ctcttaaggc tagagtactt aatacgactc actataggct agccaccatg
      961 gcttccaagg tgtacgaccc cgagcaacgc aaacgcatga tcactgggcc tcagtggtgg
     1021 gctcgctgca agcaaatgaa cgtgctggac tccttcatca actactatga ttccgagaag
     1081 cacgccgaga acgccgtgat ttttctgcat ggtaacgctg cctccagcta cctgtggagg
     1141 cacgtcgtgc ctcacatcga gcccgtggct agatgcatca tccctgatct gatcggaatg
     1201 ggtaagtccg gcaagagcgg gaatggctca tatcgcctcc tggatcacta caagtacctc
     1261 accgcttggt tcgagctgct gaaccttcca aagaaaatca tctttgtggg ccacgactgg
     1321 ggggcttgtc tggcctttca ctactcctac gagcaccaag acaagatcaa ggccatcgtc
     1381 catgctgaga gtgtcgtgga cgtgatcgag tcctgggacg agtggcctga catcgaggag
     1441 gatatcgccc tgatcaagag cgaagagggc gagaaaatgg tgcttgagaa taacttcttc
     1501 gtcgagacca tgctcccaag caagatcatg cggaaactgg agcctgagga gttcgctgcc
     1561 tacctggagc cattcaagga gaagggcgag gttagacggc ctaccctctc ctggcctcgc
     1621 gagatccctc tcgttaaggg aggcaagccc gacgtcgtcc agattgtccg caactacaac
     1681 gcctaccttc gggccagcga cgatctgcct aagatgttca tcgagtccga ccctgggttc
     1741 ttttccaacg ctattgtcga gggagctaag aagttcccta acaccgagtt cgtgaaggtg
     1801 aagggcctcc acttcagcca ggaggacgct ccagatgaaa tgggtaagta catcaagagc
     1861 ttcgtggagc gcgtgctgaa gaacgagcag taattctagc cgactcacta tagggcgaat
     1921 tggcggaagg ccgtcaaggc cgcatcaacg agctctctag actgtagaaa acatgaggat
     1981 cacccatgtc tgctggacga ctgtagaaaa catgaggatc acccatgtct gctgtctagc
     2041 tgtagaaaac atgaggatca cccatgtctg ctggacgact gtagaaaaca tgaggatcac
     2101 ccatgtctgc tgtctagctg tagaaaacat gaggatcacc catgtctgct ggacgactgt
     2161 agaaaacatg aggatcaccc atgtctgctg ggccccaaca attgcattca ttttatgttt
     2221 caggttcagg gggaggtgtg ggaggttttt taaagcaagt aaaacctcta caaatgtggt
     2281 atggctgatt atgatcagtc gaccgatgcc cttgagagcc ttcaacccag tcagctcctt
     2341 ccggtgggcg cggggcatga ctatcgtcgc cgcacttatg actgtcttct ttatcatgca
     2401 actcgtagga caggtgccgg cagcgctctg ggtcattttc ggcgaggacc gctctcgagg
     2461 cggccgcttc cctttagtga gggttaatgc ttcgagcaga catgataaga tacattgatg
     2521 agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg
     2581 atgctattgc tttatttgta accattataa gctgcaataa acaagttaac aacaacaatt
     2641 gcattcattt tatgtttcag gttcaggggg agatgtggga ggttttttaa agcaagtaaa
     2701 acctctacaa atgtggtaaa atccgataag gatcgatccg ggctggcgta atagcgaaga
     2761 ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggacgcgccc
     2821 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     2881 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     2941 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     3001 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc
     3061 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     3121 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt
     3181 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     3241 tttaacaaaa tattaacgct tacaatttcc tgatgcggta ttttctcctt acgcatctgt
     3301 gcggtatttc acaccgcata cgcggatctg cgcagcacca tggcctgaaa taacctctga
     3361 aagaggaact tggttaggta ccttctgagg cggaaagaac cagctgtgga atgtgtgtca
     3421 gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     3481 caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     3541 aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc
     3601 cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt tttttattta
     3661 tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt
     3721 tggaggccta ggcttttgca aaaagcttga ttcttctgac acaacagtct cgaacttaag
     3781 gctagagcca ccatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg
     3841 gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg
     3901 ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc
     3961 ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct
     4021 tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa
     4081 gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg
     4141 gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa
     4201 gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat
     4261 gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg
     4321 cgcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc
     4381 atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac
     4441 cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg
     4501 gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc
     4561 tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg accgaccaag
     4621 cgacgcccaa cctgccatca cgatggccgc aataaaatat ctttattttc attacatctg
     4681 tgtgttggtt ttttgtgtga atcgatagcg ataaggatcc gcgtatggtg cactctcagt
     4741 acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac
     4801 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc
     4861 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc
     4921 ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca
     4981 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     5041 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     5101 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     5161 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     5221 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     5281 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     5341 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     5401 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     5461 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     5521 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     5581 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     5641 accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     5701 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     5761 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     5821 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     5881 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     5941 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     6001 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     6061 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     6121 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     6181 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     6241 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag
     6301 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     6361 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     6421 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     6481 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     6541 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     6601 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     6661 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     6721 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     6781 gctcacatgg ctcgacagat cttcaatatt ggccattagc catattattc attggttata
     6841 tagcataaat caatattggc tattggccat tgcatacgtt gtatctatat cataatatgt
     6901 acatttatat tggctcatgt ccaatatgac cgccatgtt
//
