LOCUS       sequence-288536-        6600 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian Expression of 5BoxB.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6600)
  AUTHORS   Lazzaretti D, Tournier I, Izaurralde E
  TITLE     The C-terminal domains of human TNRC6A, TNRC6B, and TNRC6C silence 
            bound transcripts independently of Argonaute proteins.
  JOURNAL   RNA. 2009 Jun . 15(6):1059-66.
  PUBMED    19383768
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. 2009 
            Jun . 15(6):1059-66."
COMMENT     Sequence Label: pCIneo-RLuc-5BoxB_C
FEATURES             Location/Qualifiers
     source          1..6600
                     /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"
     intron          890..1022
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     1005..1024
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        1067..1085
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1089..1098
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1095..2030
                     /codon_start=1
                     /product="Renilla luciferase"
                     /label=hRluc
                     /note="human codon-optimized"
                     /translation="MASKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAEN
                     AVIFLHGNAASSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAW
                     FELLNLPKKIIFVGHDWGACLAFHYSYEHQDKIKAIVHAESVVDVIESWDEWPDIEEDI
                     ALIKSEEGEKMVLENNFFVETMLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPRE
                     IPLVKGGKPDVVQIVRNYNAYLRASDDLPKMFIESDPGFFSNAIVEGAKKFPNTEFVKV
                     KGLHFSQEDAPDEMGKYIKSFVERVLKNEQ"
     protein_bind    2067..2085
                     /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    2111..2129
                     /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    2135..2153
                     /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    2194..2212
                     /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    2218..2236
                     /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    2304..2425
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2315..2334)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2369..2388
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      2611..3066
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2698..2717)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2908..2929
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3183..3540
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3183..3374
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3206..3226)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3391..3526
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3591..4385
                     /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(3645..3664)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4255..4274
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4449..4497
                     /note="synthetic polyadenylation signal"
     primer_bind     complement(4538..4557)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4657..4679
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4717..4735)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4803..4907
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4908..4976,4977..5768)
                     /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(5126..5145)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5939..6527
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6428..6447
                     /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 actagaagct ttattgcggt agtttatcac
      781 agttaaattg ctaacgcagt cagtgcttct gacacaacag tctcgaactt aagctgcagt
      841 gactctctta aggtagcctt gcagaagttg gtcgtgaggc actgggcagg taagtatcaa
      901 ggttacaaga caggtttaag gagaccaata gaaactgggc ttgtcgagac agagaagact
      961 cttgcgtttc tgataggcac ctattggtct tactgacatc cactttgcct ttctctccac
     1021 aggtgtccac tcccagttca attacagctc ttaaggctag agtacttaat acgactcact
     1081 ataggctagc caccatggct tccaaggtgt acgaccccga gcaacgcaaa cgcatgatca
     1141 ctgggcctca gtggtgggct cgctgcaagc aaatgaacgt gctggactcc ttcatcaact
     1201 actatgattc cgagaagcac gccgagaacg ccgtgatttt tctgcatggt aacgctgcct
     1261 ccagctacct gtggaggcac gtcgtgcctc acatcgagcc cgtggctaga tgcatcatcc
     1321 ctgatctgat cggaatgggt aagtccggca agagcgggaa tggctcatat cgcctcctgg
     1381 atcactacaa gtacctcacc gcttggttcg agctgctgaa ccttccaaag aaaatcatct
     1441 ttgtgggcca cgactggggg gcttgtctgg cctttcacta ctcctacgag caccaagaca
     1501 agatcaaggc catcgtccat gctgagagtg tcgtggacgt gatcgagtcc tgggacgagt
     1561 ggcctgacat cgaggaggat atcgccctga tcaagagcga agagggcgag aaaatggtgc
     1621 ttgagaataa cttcttcgtc gagaccatgc tcccaagcaa gatcatgcgg aaactggagc
     1681 ctgaggagtt cgctgcctac ctggagccat tcaaggagaa gggcgaggtt agacggccta
     1741 ccctctcctg gcctcgcgag atccctctcg ttaagggagg caagcccgac gtcgtccaga
     1801 ttgtccgcaa ctacaacgcc taccttcggg ccagcgacga tctgcctaag atgttcatcg
     1861 agtccgaccc tgggttcttt tccaacgcta ttgtcgaggg agctaagaag ttccctaaca
     1921 ccgagttcgt gaaggtgaag ggcctccact tcagccagga ggacgctcca gatgaaatgg
     1981 gtaagtacat caagagcttc gtggagcgcg tgctgaagaa cgagcagtaa ttctagataa
     2041 gtccaactac taaactgggg attcctgggc cctgaagaag ggcccctcga ctaagtccaa
     2101 ctactaaact gggccctgaa gaagggccca tatagggccc tgaagaaggg ccctatcgag
     2161 gatattatct cgactaagtc caactactaa actgggccct gaagaagggc ccatataggg
     2221 ccctgaagaa gggccctatc gaggatatta tctcgaggcg gccgcttccc tttagtgagg
     2281 gttaatgctt cgagcagaca tgataagata cattgatgag tttggacaaa ccacaactag
     2341 aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac
     2401 cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta tgtttcaggt
     2461 tcagggggag atgtgggagg ttttttaaag caagtaaaac ctctacaaat gtggtaaaat
     2521 ccgataagga tcgatccggg ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc
     2581 caacagttgc gcagcctgaa tggcgaatgg acgcgccctg tagcggcgca ttaagcgcgg
     2641 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
     2701 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     2761 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     2821 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     2881 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     2941 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
     3001 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta
     3061 caatttcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatacg
     3121 cggatctgcg cagcaccatg gcctgaaata acctctgaaa gaggaacttg gttaggtacc
     3181 ttctgaggcg gaaagaacca gctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca
     3241 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt
     3301 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     3361 gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
     3421 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg
     3481 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa
     3541 aagcttgatt cttctgacac aacagtctcg aacttaaggc tagagccacc atgattgaac
     3601 aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact
     3661 gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc
     3721 gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg caggacgagg
     3781 cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg
     3841 tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt
     3901 catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc
     3961 atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag
     4021 cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg
     4081 ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc
     4141 tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt
     4201 ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg
     4261 ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt
     4321 acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct
     4381 tctgagcggg actctggggt tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg
     4441 atggccgcaa taaaatatct ttattttcat tacatctgtg tgttggtttt ttgtgtgaat
     4501 cgatagcgat aaggatccgc gtatggtgca ctctcagtac aatctgctct gatgccgcat
     4561 agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
     4621 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
     4681 tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat
     4741 aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg
     4801 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     4861 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     4921 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     4981 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     5041 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     5101 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg
     5161 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     5221 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     5281 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     5341 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     5401 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg
     5461 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     5521 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     5581 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg
     5641 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     5701 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     5761 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     5821 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     5881 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     5941 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     6001 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     6061 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
     6121 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     6181 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     6241 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     6301 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     6361 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     6421 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     6481 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     6541 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatggct cgacagatct
//
