LOCUS       sequence-290883-        6605 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Mammalian Expression of 6xbsMS2-8xlet7.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6605)
  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 6605)
  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"; 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-6xbsMS2-8xlet7_AC
FEATURES             Location/Qualifiers
     source          1..6605
                     /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    2309..2430
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2320..2339)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2374..2393
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      2616..3071
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2703..2722)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2913..2934
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3188..3545
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3188..3379
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3211..3231)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3396..3531
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3596..4390
                     /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(3650..3669)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4260..4279
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4454..4502
                     /note="synthetic polyadenylation signal"
     primer_bind     complement(4543..4562)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4662..4684
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4722..4740)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4808..4912
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4913..4981,4982..5773)
                     /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(5131..5150)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5944..6532
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6433..6452
                     /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 ctgggcccgc ttcgtggttc tggcgatcga tcgagactat acaaggatct
     2041 acctcagtcg agactataca aggatctacc tcagtcgaga ctatacaagg atctacctca
     2101 gtcgagacta tacaaggatc tacctcagtc gagactatac aaggatctac ctcagtcgag
     2161 actatacaag gatctacctc agtcgagact atacaaggat ctacctcagt cgagactata
     2221 caaggatcta cctcagtcga cgcggccgct ggccgcctcg aggcggccgc ttccctttag
     2281 tgagggttaa tgcttcgagc agacatgata agatacattg atgagtttgg acaaaccaca
     2341 actagaatgc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat tgctttattt
     2401 gtaaccatta taagctgcaa taaacaagtt aacaacaaca attgcattca ttttatgttt
     2461 caggttcagg gggagatgtg ggaggttttt taaagcaagt aaaacctcta caaatgtggt
     2521 aaaatccgat aaggatcgat ccgggctggc gtaatagcga agaggcccgc accgatcgcc
     2581 cttcccaaca gttgcgcagc ctgaatggcg aatggacgcg ccctgtagcg gcgcattaag
     2641 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     2701 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     2761 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     2821 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     2881 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     2941 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
     3001 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
     3061 gcttacaatt tcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     3121 atacgcggat ctgcgcagca ccatggcctg aaataacctc tgaaagagga acttggttag
     3181 gtaccttctg aggcggaaag aaccagctgt ggaatgtgtg tcagttaggg tgtggaaagt
     3241 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     3301 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     3361 agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
     3421 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
     3481 cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt
     3541 gcaaaaagct tgattcttct gacacaacag tctcgaactt aaggctagag ccaccatgat
     3601 tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta
     3661 tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca
     3721 ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcagga
     3781 cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga
     3841 cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct
     3901 cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg
     3961 gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga
     4021 gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca
     4081 tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgcgcatgc ccgacggcga
     4141 ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg
     4201 cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc
     4261 gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt
     4321 gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga
     4381 gttcttctga gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca
     4441 tcacgatggc cgcaataaaa tatctttatt ttcattacat ctgtgtgttg gttttttgtg
     4501 tgaatcgata gcgataagga tccgcgtatg gtgcactctc agtacaatct gctctgatgc
     4561 cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
     4621 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
     4681 gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt
     4741 tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg
     4801 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     4861 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     4921 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     4981 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     5041 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     5101 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
     5161 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     5221 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     5281 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     5341 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     5401 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
     5461 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     5521 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     5581 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
     5641 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     5701 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     5761 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     5821 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     5881 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     5941 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     6001 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     6061 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     6121 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     6181 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     6241 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     6301 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     6361 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     6421 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     6481 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     6541 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tggctcgaca
     6601 gatct
//
