LOCUS       sequence-290882-        6405 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian Expression of 6xbsMS2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6405)
  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 6405)
  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-TISU-RLuc-6xbsMS2_AC
FEATURES             Location/Qualifiers
     source          1..6405
                     /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"
     CDS             764..1696
                     /codon_start=1
                     /product="Renilla luciferase"
                     /label=hRluc
                     /note="human codon-optimized"
                     /translation="ASKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAENA
                     VIFLHGNAASSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAWF
                     ELLNLPKKIIFVGHDWGACLAFHYSYEHQDKIKAIVHAESVVDVIESWDEWPDIEEDIA
                     LIKSEEGEKMVLENNFFVETMLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPREI
                     PLVKGGKPDVVQIVRNYNAYLRASDDLPKMFIESDPGFFSNAIVEGAKKFPNTEFVKVK
                     GLHFSQEDAPDEMGKYIKSFVERVLKNEQ"
     misc_RNA        1774..1792
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        1813..1831
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        1852..1870
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        1891..1909
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        1930..1948
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        1969..1987
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     polyA_signal    2109..2230
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2120..2139)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2174..2193
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      2416..2871
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2503..2522)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2713..2734
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2988..3345
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2988..3179
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3011..3031)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3196..3331
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3396..4190
                     /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(3450..3469)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4060..4079
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4254..4302
                     /note="synthetic polyadenylation signal"
     primer_bind     complement(4343..4362)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4462..4484
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4522..4540)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4608..4712
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4713..4781,4782..5573)
                     /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(4931..4950)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5744..6332
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6233..6252
                     /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 caagatggcg gcagcttcca aggtgtacga
      781 ccccgagcaa cgcaaacgca tgatcactgg gcctcagtgg tgggctcgct gcaagcaaat
      841 gaacgtgctg gactccttca tcaactacta tgattccgag aagcacgccg agaacgccgt
      901 gatttttctg catggtaacg ctgcctccag ctacctgtgg aggcacgtcg tgcctcacat
      961 cgagcccgtg gctagatgca tcatccctga tctgatcgga atgggtaagt ccggcaagag
     1021 cgggaatggc tcatatcgcc tcctggatca ctacaagtac ctcaccgctt ggttcgagct
     1081 gctgaacctt ccaaagaaaa tcatctttgt gggccacgac tggggggctt gtctggcctt
     1141 tcactactcc tacgagcacc aagacaagat caaggccatc gtccatgctg agagtgtcgt
     1201 ggacgtgatc gagtcctggg acgagtggcc tgacatcgag gaggatatcg ccctgatcaa
     1261 gagcgaagag ggcgagaaaa tggtgcttga gaataacttc ttcgtcgaga ccatgctccc
     1321 aagcaagatc atgcggaaac tggagcctga ggagttcgct gcctacctgg agccattcaa
     1381 ggagaagggc gaggttagac ggcctaccct ctcctggcct cgcgagatcc ctctcgttaa
     1441 gggaggcaag cccgacgtcg tccagattgt ccgcaactac aacgcctacc ttcgggccag
     1501 cgacgatctg cctaagatgt tcatcgagtc cgaccctggg ttcttttcca acgctattgt
     1561 cgagggagct aagaagttcc ctaacaccga gttcgtgaag gtgaagggcc tccacttcag
     1621 ccaggaggac gctccagatg aaatgggtaa gtacatcaag agcttcgtgg agcgcgtgct
     1681 gaagaacgag cagtaattct agccgactca ctatagggcg aattggcgga aggccgtcaa
     1741 ggccgcatca acgagctctc tagactgtag aaaacatgag gatcacccat gtctgctgga
     1801 cgactgtaga aaacatgagg atcacccatg tctgctgtct agctgtagaa aacatgagga
     1861 tcacccatgt ctgctggacg actgtagaaa acatgaggat cacccatgtc tgctgtctag
     1921 ctgtagaaaa catgaggatc acccatgtct gctggacgac tgtagaaaac atgaggatca
     1981 cccatgtctg ctgggcccgg taccctctgg gcctcatggg ccttccgctc actgcccgct
     2041 ttccagtcgg gaaaccctcg aggcggccgc ttccctttag tgagggttaa tgcttcgagc
     2101 agacatgata agatacattg atgagtttgg acaaaccaca actagaatgc agtgaaaaaa
     2161 atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa
     2221 taaacaagtt aacaacaaca attgcattca ttttatgttt caggttcagg gggagatgtg
     2281 ggaggttttt taaagcaagt aaaacctcta caaatgtggt aaaatccgat aaggatcgat
     2341 ccgggctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc
     2401 ctgaatggcg aatggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     2461 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     2521 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     2581 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     2641 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     2701 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     2761 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     2821 tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt tcctgatgcg
     2881 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atacgcggat ctgcgcagca
     2941 ccatggcctg aaataacctc tgaaagagga acttggttag gtaccttctg aggcggaaag
     3001 aaccagctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc
     3061 agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc
     3121 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg
     3181 cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat
     3241 ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc
     3301 cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct tgattcttct
     3361 gacacaacag tctcgaactt aaggctagag ccaccatgat tgaacaagat ggattgcacg
     3421 caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa
     3481 tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg
     3541 tcaagaccga cctgtccggt gccctgaatg aactgcagga cgaggcagcg cggctatcgt
     3601 ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa
     3661 gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc
     3721 ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg
     3781 ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg
     3841 aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg
     3901 aactgttcgc caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg
     3961 gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact
     4021 gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg
     4081 ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc
     4141 ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct
     4201 ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgatggc cgcaataaaa
     4261 tatctttatt ttcattacat ctgtgtgttg gttttttgtg tgaatcgata gcgataagga
     4321 tccgcgtatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc
     4381 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     4441 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     4501 cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga
     4561 taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     4621 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     4681 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     4741 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     4801 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     4861 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     4921 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
     4981 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     5041 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     5101 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     5161 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     5221 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
     5281 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     5341 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     5401 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
     5461 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     5521 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     5581 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     5641 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     5701 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     5761 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     5821 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     5881 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     5941 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     6001 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     6061 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     6121 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     6181 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     6241 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     6301 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     6361 tcctggcctt ttgctggcct tttgctcaca tggctcgaca gatct
//
