LOCUS       sequence-223057-        6600 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression vector to produce humanized Renilla luciferase with 5BoxB
            sequences in the 3'UTR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6600)
  AUTHORS   Pillai RS, Artus CG, Filipowicz W
  TITLE     Tethering of human Ago proteins to mRNA mimics the miRNA-mediated 
            repression of protein synthesis.
  JOURNAL   RNA. 2004 Oct . 10(10):1518-25.
  PUBMED    15337849
REFERENCE   2  (bases 1 to 6600)
  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"; journalName: "RNA. 2004 
            Oct . 10(10):1518-25."
COMMENT     Sequence Label: pCIneo-RL-5BoxB
FEATURES             Location/Qualifiers
     source          1..6600
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        800..1179
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1180..1391
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1342..1362
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1388..1412
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          1552..1684
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     1667..1686
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        1729..1747
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1751..1760
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1757..2692
                     /codon_start=1
                     /product="Renilla luciferase"
                     /label=hRluc
                     /note="human codon-optimized"
                     /translation="MASKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAEN
                     AVIFLHGNAASSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAW
                     FELLNLPKKIIFVGHDWGACLAFHYSYEHQDKIKAIVHAESVVDVIESWDEWPDIEEDI
                     ALIKSEEGEKMVLENNFFVETMLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPRE
                     IPLVKGGKPDVVQIVRNYNAYLRASDDLPKMFIESDPGFFSNAIVEGAKKFPNTEFVKV
                     KGLHFSQEDAPDEMGKYIKSFVERVLKNEQ"
     protein_bind    2729..2747
                     /label=boxB
                     /bound_moiety="lambda-N peptide"
                     /note="lambda N peptide binding site consisting of an RNA 
                     hairpin within the N-utliization (nut) site of the lambda 
                     bacteriophage transcript (Tan and Frankel, 1995)"
     protein_bind    2773..2791
                     /label=boxB
                     /bound_moiety="lambda-N peptide"
                     /note="lambda N peptide binding site consisting of an RNA 
                     hairpin within the N-utliization (nut) site of the lambda 
                     bacteriophage transcript (Tan and Frankel, 1995)"
     protein_bind    2797..2815
                     /label=boxB
                     /bound_moiety="lambda-N peptide"
                     /note="lambda N peptide binding site consisting of an RNA 
                     hairpin within the N-utliization (nut) site of the lambda 
                     bacteriophage transcript (Tan and Frankel, 1995)"
     protein_bind    2856..2874
                     /label=boxB
                     /bound_moiety="lambda-N peptide"
                     /note="lambda N peptide binding site consisting of an RNA 
                     hairpin within the N-utliization (nut) site of the lambda 
                     bacteriophage transcript (Tan and Frankel, 1995)"
     protein_bind    2880..2898
                     /label=boxB
                     /bound_moiety="lambda-N peptide"
                     /note="lambda N peptide binding site consisting of an RNA 
                     hairpin within the N-utliization (nut) site of the lambda 
                     bacteriophage transcript (Tan and Frankel, 1995)"
     polyA_signal    2966..3087
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2977..2996)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3031..3050
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      3273..3728
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3360..3379)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3570..3591
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3845..4202
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3845..4036
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3868..3888)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4053..4188
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4253..5047
                     /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(4307..4326)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4917..4936
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5111..5159
                     /note="synthetic polyadenylation signal"
     primer_bind     complement(5200..5219)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5319..5341
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5379..5397)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5465..5569
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5570..5638,5639..6430)
                     /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(5788..5807)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgg ctcgacagat
      661 cttcaatatt ggccattagc catattattc attggttata tagcataaat caatattggc
      721 tattggccat tgcatacgtt gtatctatat cataatatgt acatttatat tggctcatgt
      781 ccaatatgac cgccatgttg gcattgatta ttgactagtt attaatagta atcaattacg
      841 gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc
      901 ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc
      961 atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact
     1021 gcccacttgg cagtacatca agtgtatcat atgccaagtc cgccccctat tgacgtcaat
     1081 gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttacggga ctttcctact
     1141 tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac
     1201 accaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac
     1261 gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac
     1321 tgcgatcgcc cgccccgttg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata
     1381 taagcagagc tcgtttagtg aaccgtcaga tcactagaag ctttattgcg gtagtttatc
     1441 acagttaaat tgctaacgca gtcagtgctt ctgacacaac agtctcgaac ttaagctgca
     1501 gtgactctct taaggtagcc ttgcagaagt tggtcgtgag gcactgggca ggtaagtatc
     1561 aaggttacaa gacaggttta aggagaccaa tagaaactgg gcttgtcgag acagagaaga
     1621 ctcttgcgtt tctgataggc acctattggt cttactgaca tccactttgc ctttctctcc
     1681 acaggtgtcc actcccagtt caattacagc tcttaaggct agagtactta atacgactca
     1741 ctataggcta gccaccatgg cttccaaggt gtacgacccc gagcaacgca aacgcatgat
     1801 cactgggcct cagtggtggg ctcgctgcaa gcaaatgaac gtgctggact ccttcatcaa
     1861 ctactatgat tccgagaagc acgccgagaa cgccgtgatt tttctgcatg gtaacgctgc
     1921 ctccagctac ctgtggaggc acgtcgtgcc tcacatcgag cccgtggcta gatgcatcat
     1981 ccctgatctg atcggaatgg gtaagtccgg caagagcggg aatggctcat atcgcctcct
     2041 ggatcactac aagtacctca ccgcttggtt cgagctgctg aaccttccaa agaaaatcat
     2101 ctttgtgggc cacgactggg gggcttgtct ggcctttcac tactcctacg agcaccaaga
     2161 caagatcaag gccatcgtcc atgctgagag tgtcgtggac gtgatcgagt cctgggacga
     2221 gtggcctgac atcgaggagg atatcgccct gatcaagagc gaagagggcg agaaaatggt
     2281 gcttgagaat aacttcttcg tcgagaccat gctcccaagc aagatcatgc ggaaactgga
     2341 gcctgaggag ttcgctgcct acctggagcc attcaaggag aagggcgagg ttagacggcc
     2401 taccctctcc tggcctcgcg agatccctct cgttaaggga ggcaagcccg acgtcgtcca
     2461 gattgtccgc aactacaacg cctaccttcg ggccagcgac gatctgccta agatgttcat
     2521 cgagtccgac cctgggttct tttccaacgc tattgtcgag ggagctaaga agttccctaa
     2581 caccgagttc gtgaaggtga agggcctcca cttcagccag gaggacgctc cagatgaaat
     2641 gggtaagtac atcaagagct tcgtggagcg cgtgctgaag aacgagcagt aattctagat
     2701 aagtccaact actaaactgg ggattcctgg gccctgaaga agggcccctc gactaagtcc
     2761 aactactaaa ctgggccctg aagaagggcc catatagggc cctgaagaag ggccctatcg
     2821 aggatattat ctcgactaag tccaactact aaactgggcc ctgaagaagg gcccatatag
     2881 ggccctgaag aagggcccta tcgaggatat tatctcgagg cggccgcttc cctttagtga
     2941 gggttaatgc ttcgagcaga catgataaga tacattgatg agtttggaca aaccacaact
     3001 agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta
     3061 accattataa gctgcaataa acaagttaac aacaacaatt gcattcattt tatgtttcag
     3121 gttcaggggg agatgtggga ggttttttaa agcaagtaaa acctctacaa atgtggtaaa
     3181 atccgataag gatcgatccg ggctggcgta atagcgaaga ggcccgcacc gatcgccctt
     3241 cccaacagtt gcgcagcctg aatggcgaat ggacgcgccc tgtagcggcg cattaagcgc
     3301 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     3361 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     3421 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     3481 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     3541 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     3601 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     3661 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct
     3721 tacaatttcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
     3781 cgcggatctg cgcagcacca tggcctgaaa taacctctga aagaggaact tggttaggta
     3841 ccttctgagg cggaaagaac cagctgtgga atgtgtgtca gttagggtgt ggaaagtccc
     3901 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt
     3961 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     4021 cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg
     4081 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct
     4141 cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca
     4201 aaaagcttga ttcttctgac acaacagtct cgaacttaag gctagagcca ccatgattga
     4261 acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga
     4321 ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg
     4381 gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaggacga
     4441 ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt
     4501 tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct
     4561 gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct
     4621 gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg
     4681 agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca
     4741 ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg acggcgagga
     4801 tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt
     4861 ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt
     4921 ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct
     4981 ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt
     5041 cttctgagcg ggactctggg gttcgaaatg accgaccaag cgacgcccaa cctgccatca
     5101 cgatggccgc aataaaatat ctttattttc attacatctg tgtgttggtt ttttgtgtga
     5161 atcgatagcg ataaggatcc gcgtatggtg cactctcagt acaatctgct ctgatgccgc
     5221 atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     5281 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
     5341 gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt
     5401 ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa
     5461 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     5521 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
     5581 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     5641 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     5701 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     5761 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc
     5821 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     5881 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     5941 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     6001 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     6061 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat
     6121 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
     6181 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     6241 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat
     6301 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     6361 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     6421 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
     6481 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     6541 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
//
