LOCUS       sequence-223055-        6465 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression vector to produce humanized Renilla luciferase with three
            mutated (seed-sequence mutations) partially complementary binding 
            sites for human let-7 miRNA in the 3'UTR .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6465)
  AUTHORS   Pillai RS, Bhattacharyya SN, Artus CG, Zoller T, Cougot N, Basyuk E,
            Bertrand E, Filipowicz W
  TITLE     Inhibition of translational initiation by Let-7 MicroRNA in human 
            cells.
  JOURNAL   Science. 2005 Sep 2. 309(5740):1573-6.
  PUBMED    16081698
REFERENCE   2  (bases 1 to 6465)
  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: "Science. 
            2005 Sep 2. 309(5740):1573-6."
COMMENT     Sequence Label: p CIneo-RL-Let7-3xBulgeB-mut
FEATURES             Location/Qualifiers
     source          1..6465
                     /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"
     polyA_signal    2831..2952
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2842..2861)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2896..2915
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      3138..3593
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3225..3244)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3435..3456
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3710..4067
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3710..3901
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3733..3753)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3918..4053
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4118..4912
                     /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(4172..4191)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4782..4801
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4976..5024
                     /note="synthetic polyadenylation signal"
     primer_bind     complement(5065..5084)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5184..5206
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5244..5262)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5330..5434
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5435..5503,5504..6295)
                     /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(5653..5672)
                     /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 aatctagagc
     2701 acagcctatt gaactacccc tcactcgagc acagcctatt gaaactaccc ctcaggcctg
     2761 cacagcctat tgaactaccc ctcagcggcc gcttcccttt agtgagggtt aatgcttcga
     2821 gcagacatga taagatacat tgatgagttt ggacaaacca caactagaat gcagtgaaaa
     2881 aaatgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc
     2941 aataaacaag ttaacaacaa caattgcatt cattttatgt ttcaggttca gggggagatg
     3001 tgggaggttt tttaaagcaa gtaaaacctc tacaaatgtg gtaaaatccg ataaggatcg
     3061 atccgggctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca
     3121 gcctgaatgg cgaatggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     3181 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     3241 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     3301 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     3361 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     3421 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     3481 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     3541 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa tttcctgatg
     3601 cggtattttc tccttacgca tctgtgcggt atttcacacc gcatacgcgg atctgcgcag
     3661 caccatggcc tgaaataacc tctgaaagag gaacttggtt aggtaccttc tgaggcggaa
     3721 agaaccagct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag
     3781 gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag
     3841 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc
     3901 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc
     3961 atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat
     4021 tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag cttgattctt
     4081 ctgacacaac agtctcgaac ttaaggctag agccaccatg attgaacaag atggattgca
     4141 cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac
     4201 aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt
     4261 tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc
     4321 gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg
     4381 aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc
     4441 tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc
     4501 ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat
     4561 ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc
     4621 cgaactgttc gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca
     4681 tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga
     4741 ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat
     4801 tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc
     4861 tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact
     4921 ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgatg gccgcaataa
     4981 aatatcttta ttttcattac atctgtgtgt tggttttttg tgtgaatcga tagcgataag
     5041 gatccgcgta tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc
     5101 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
     5161 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc
     5221 accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat
     5281 gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     5341 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     5401 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     5461 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     5521 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     5581 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     5641 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
     5701 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     5761 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     5821 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     5881 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     5941 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg
     6001 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     6061 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     6121 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc
     6181 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     6241 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     6301 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     6361 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     6421 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttc
//
