LOCUS       sequence-223053-        6459 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression vector to produce humanized Renilla luciferase with three
            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 6459)
  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 6459)
  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
FEATURES             Location/Qualifiers
     source          1..6459
                     /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    2825..2946
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2836..2855)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2890..2909
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      3132..3587
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3219..3238)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3429..3450
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3704..4061
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3704..3895
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3727..3747)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3912..4047
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4112..4906
                     /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(4166..4185)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4776..4795
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4970..5018
                     /note="synthetic polyadenylation signal"
     primer_bind     complement(5059..5078)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5178..5200
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5238..5256)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5324..5428
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5429..5497,5498..6289)
                     /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(5647..5666)
                     /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 aatctagagg
     2701 acagcctatt gaactacctc actcggagca cagcctattg aactacctca ggcctgcaca
     2761 gcctattgaa ctacctcagc ggccgcttcc ctttagtgag ggttaatgct tcgagcagac
     2821 atgataagat acattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc
     2881 tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa
     2941 caagttaaca acaacaattg cattcatttt atgtttcagg ttcaggggga gatgtgggag
     3001 gttttttaaa gcaagtaaaa cctctacaaa tgtggtaaaa tccgataagg atcgatccgg
     3061 gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga
     3121 atggcgaatg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     3181 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     3241 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     3301 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     3361 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     3421 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     3481 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     3541 acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttcct gatgcggtat
     3601 tttctcctta cgcatctgtg cggtatttca caccgcatac gcggatctgc gcagcaccat
     3661 ggcctgaaat aacctctgaa agaggaactt ggttaggtac cttctgaggc ggaaagaacc
     3721 agctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa
     3781 gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc
     3841 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc
     3901 taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct
     3961 gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga
     4021 agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagcttgat tcttctgaca
     4081 caacagtctc gaacttaagg ctagagccac catgattgaa caagatggat tgcacgcagg
     4141 ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg
     4201 ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa
     4261 gaccgacctg tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct
     4321 ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga
     4381 ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc
     4441 cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac
     4501 ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc
     4561 cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact
     4621 gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga
     4681 tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg
     4741 ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga
     4801 agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga
     4861 ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg
     4921 ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gatggccgca ataaaatatc
     4981 tttattttca ttacatctgt gtgttggttt tttgtgtgaa tcgatagcga taaggatccg
     5041 cgtatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca
     5101 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     5161 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     5221 acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat
     5281 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     5341 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     5401 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     5461 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     5521 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     5581 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     5641 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     5701 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     5761 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     5821 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     5881 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     5941 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
     6001 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     6061 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     6121 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
     6181 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     6241 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     6301 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     6361 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     6421 ctgagcgtca gaccccgtag aaaagatcaa aggatcttc
//
