LOCUS       sequence-474566-        8024 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian Expression of 6xbsMS2-Malat.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8024)
  AUTHORS   Bhandari D, Raisch T, Weichenrieder O, Jonas S, Izaurralde E
  TITLE     Structural basis for the Nanos-mediated recruitment of the CCR4-NOT 
            complex and translational repression.
  JOURNAL   Genes Dev. 2014 Apr 15;28(8):888-901. doi: 10.1101/gad.237289.113.
  PUBMED    24736845
REFERENCE   2  (bases 1 to 8024)
  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: 
            "10.1101/gad.237289"; journalName: "Genes Dev"; date: "2014-04-15- 
            15"; volume: "28"; issue: "8"; pages: "888-901"
COMMENT     Sequence Label: pcDNA3.1-beta-hRLuc-V5-6xbsMS2-MALAT_X
FEATURES             Location/Qualifiers
     source          1..8024
                     /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"
     CDS             2367..3299
                     /codon_start=1
                     /product="Renilla luciferase"
                     /label=hRluc
                     /note="human codon-optimized"
                     /translation="MASKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAEN
                     AVIFLHGNAASSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAW
                     FELLNLPKKIIFVGHDWGACLAFHYSYEHQDKIKAIVHAESVVDVIESWDEWPDIEEDI
                     ALIKSEEGEKMVLENNFFVETMLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPRE
                     IPLVKGGKPDVVQIVRNYNAYLRASDDLPKMFIESDPGFFSNAIVEGAKKFPNTEFVKV
                     KGLHFSQEDAPDEMGKYIKSFVERVLKNEQ"
     CDS             3300..3341
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     misc_RNA        3441..3459
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        3480..3498
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        3519..3537
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        3558..3576
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        3597..3615
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        3636..3654
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     rep_origin      3892..4320
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3979..3998)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4189..4210
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4329..4349)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4334..4663
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4514..4649
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4576..4595
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4730..5524
                     /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(4784..4803)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5394..5413
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5698..5819
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5735..5754)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5789..5808
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5868..5884)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5868..5884)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5881..5903)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5892..5908
                     /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(5916..5922,5923..5940,5941..5946))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5961..5982
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6099..6116)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6270..6857)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6350..6369)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7028..7819,7820..7888))
                     /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     7651..7670
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7889..7993)
                     /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 gcttacattt
      901 gcttctgaca caactgtgtt cactagcaac ctcaaacaga caccatggtg cacctgactc
      961 ctgaggagaa gtctgccgtt actgccctgt ggggcaaggt gaacgtggat gaagttggtg
     1021 gtgaggccct gggcaggttg gtatcaaggt tacaagacag gtttaaggag accaatagaa
     1081 actgggcatg tggagacaga gaagactctt gggtttctga taggcactga ctctctctgc
     1141 ctattggtct attttcccac ccttaggctg ctggtggtct acccttggac ccagaggttc
     1201 tttgagtcct ttggggatct gtccactcct gatgctgtta tgggcaaccc taaggtgaag
     1261 gctcatggca agaaagtgct cggtgccttt agtgatggcc tggctcacct ggacaacctc
     1321 aagggcacct ttgccacact gagtgagctg cactgtgaca agctgcacgt ggatcctgag
     1381 aacttcaggg tgagtctatg ggacccttga tgttttcttt ccccttcttt tctatggtta
     1441 agttcatgtc ataggaaggg gataagtaac agggtacagt ttagaatggg aaacagacga
     1501 atgattgcat cagtgtggaa gtctcaggat cgttttagtt tcttttattt gctgttcata
     1561 acaattgttt tcttttgttt aattcttgct ttcttttttt ttcttctccg caatttttac
     1621 tattatactt aatgccttaa cattgtgtat aacaaaagga aatatctctg agatacatta
     1681 agtaacttaa aaaaaaaact ttacacagtc tgcctagtac attactattt ggaatatatg
     1741 tgtgcttatt tgcatattca taatctccct actttatttt cttttatttt taattgatac
     1801 ataatcatta tacatattta tgggttaaag tgtaatgttt taatatgtgt acacatattg
     1861 accaaatcag ggtaattttg catttgtaat tttaaaaaat gctttcttct tttaatatac
     1921 ttttttgttt atcttatttc taatactttc cctaatctct ttctttcagg gcaataatga
     1981 tacaatgtat catgcctctt tgcaccattc taaagaataa cagtgataat ttctgggtta
     2041 aggcaatagc aatatttctg catataaata tttctgcata taaattgtaa ctgatgtaag
     2101 aggtttcata ttgctaatag cagctacaat ccagctacca ttctgctttt attttatggt
     2161 tgggataagg ctggattatt ctgagtccaa gctaggccct tttgctaatc atgttcatac
     2221 ctcttatctt cctcccacag ctcctgggca acgtgctggt ctgtgtgctg gcccatcact
     2281 ttggcaaaga attcacccca ccagtgcagg ctgcctatca gaaagtggtg gctggtgtgg
     2341 ctaatgccct ggcccacaag tatcacatgg cttccaaggt gtacgacccc gagcaacgca
     2401 aacgcatgat cactgggcct cagtggtggg ctcgctgcaa gcaaatgaac gtgctggact
     2461 ccttcatcaa ctactatgat tccgagaagc acgccgagaa cgccgtgatt tttctgcatg
     2521 gtaacgctgc ctccagctac ctgtggaggc acgtcgtgcc tcacatcgag cccgtggcta
     2581 gatgcatcat ccctgatctg atcggaatgg gtaagtccgg caagagcggg aatggctcat
     2641 atcgcctcct ggatcactac aagtacctca ccgcttggtt cgagctgctg aaccttccaa
     2701 agaaaatcat ctttgtgggc cacgactggg gggcttgtct ggcctttcac tactcctacg
     2761 agcaccaaga caagatcaag gccatcgtcc atgctgagag tgtcgtggac gtgatcgagt
     2821 cctgggacga gtggcctgac atcgaggagg atatcgccct gatcaagagc gaagagggcg
     2881 agaaaatggt gcttgagaat aacttcttcg tcgagaccat gctcccaagc aagatcatgc
     2941 ggaaactgga gcctgaggag ttcgctgcct acctggagcc attcaaggag aagggcgagg
     3001 ttagacggcc taccctctcc tggcctcgcg agatccctct cgttaaggga ggcaagcccg
     3061 acgtcgtcca gattgtccgc aactacaacg cctaccttcg ggccagcgac gatctgccta
     3121 agatgttcat cgagtccgac cctgggttct tttccaacgc tattgtcgag ggagctaaga
     3181 agttccctaa caccgagttc gtgaaggtga agggcctcca cttcagccag gaggacgctc
     3241 cagatgaaat gggtaagtac atcaagagct tcgtggagcg cgtgctgaag aacgagcagg
     3301 gtaagcctat ccctaaccct ctcctcggtc tcgattctac gtaagcggcc gctccgcttt
     3361 cttgctgtcc aatttctatt aaaggttcct ttgttcccta agtccaacta ccaaactggg
     3421 tctagctcta gctgtagaaa acatgaggat cacccatgtc tgctggacga ctgtagaaaa
     3481 catgaggatc acccatgtct gctgtctagc tgtagaaaac atgaggatca cccatgtctg
     3541 ctggacgact gtagaaaaca tgaggatcac ccatgtctgc tgtctagctg tagaaaacat
     3601 gaggatcacc catgtctgct ggacgactgt agaaaacatg aggatcaccc atgtctgctg
     3661 tctagagatt cgtcagtagg gttgtaaagg tttttctttt cctgagaaaa caaccttttg
     3721 ttttctcagg ttttgctttt tggcctttcc ctagctttaa aaaaaaaaaa gcaaaagacg
     3781 ctggtggctg gcactcctgg tttccaggac ggggttcaag tccctgcggt gtctttgctt
     3841 ctcgagcttc tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct
     3901 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     3961 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     4021 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     4081 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
     4141 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     4201 tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt
     4261 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     4321 aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag
     4381 aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc
     4441 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc
     4501 cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg
     4561 ctgactaatt ttttttattt atgcagaggc cgaggccgcc tctgcctctg agctattcca
     4621 gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagctcc cgggagcttg
     4681 tatatccatt ttcggatctg atcaagagac aggatgagga tcgtttcgca tgattgaaca
     4741 agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg
     4801 ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg
     4861 cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aggacgaggc
     4921 agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt
     4981 cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc
     5041 atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca
     5101 tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc
     5161 acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg
     5221 gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc atgcccgacg gcgaggatct
     5281 cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc
     5341 tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc
     5401 tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta
     5461 cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt
     5521 ctgagcggga ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga
     5581 gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac
     5641 gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac
     5701 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     5761 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat
     5821 catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt
     5881 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     5941 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     6001 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     6061 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     6121 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     6181 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     6241 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     6301 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     6361 gcgtttcccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     6421 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     6481 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     6541 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     6601 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     6661 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     6721 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     6781 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     6841 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     6901 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     6961 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     7021 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     7081 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     7141 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     7201 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     7261 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     7321 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     7381 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     7441 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     7501 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     7561 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     7621 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     7681 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     7741 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     7801 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     7861 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     7921 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     7981 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtc
//
