LOCUS       sequence-223052-        6406 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression vector to produce humanized Renilla luciferase with a 
            single perfectly complementary binding site 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 6406)
  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 6406)
  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-perf
FEATURES             Location/Qualifiers
     source          1..6406
                     /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    2772..2893
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2783..2802)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2837..2856
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      3079..3534
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3166..3185)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3376..3397
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3651..4008
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3651..3842
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3674..3694)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3859..3994
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4059..4853
                     /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(4113..4132)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4723..4742
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4917..4965
                     /note="synthetic polyadenylation signal"
     primer_bind     complement(5006..5025)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5125..5147
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5185..5203)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5271..5375
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5376..5444,5445..6236)
                     /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(5594..5613)
                     /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 aattctagaa
     2701 ctatacaacc tactacctca ctcgagcggc cgcttccctt tagtgagggt taatgcttcg
     2761 agcagacatg ataagataca ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa
     2821 aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta tttgtaacca ttataagctg
     2881 caataaacaa gttaacaaca acaattgcat tcattttatg tttcaggttc agggggagat
     2941 gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt ggtaaaatcc gataaggatc
     3001 gatccgggct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc
     3061 agcctgaatg gcgaatggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     3121 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     3181 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     3241 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     3301 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     3361 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     3421 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     3481 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttcctgat
     3541 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatacgcg gatctgcgca
     3601 gcaccatggc ctgaaataac ctctgaaaga ggaacttggt taggtacctt ctgaggcgga
     3661 aagaaccagc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca
     3721 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca
     3781 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc
     3841 ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc
     3901 catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta
     3961 ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gcttgattct
     4021 tctgacacaa cagtctcgaa cttaaggcta gagccaccat gattgaacaa gatggattgc
     4081 acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga
     4141 caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt
     4201 ttgtcaagac cgacctgtcc ggtgccctga atgaactgca ggacgaggca gcgcggctat
     4261 cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg
     4321 gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg
     4381 ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc
     4441 cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga
     4501 tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag
     4561 ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc
     4621 atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg
     4681 actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata
     4741 ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg
     4801 ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac
     4861 tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgat ggccgcaata
     4921 aaatatcttt attttcatta catctgtgtg ttggtttttt gtgtgaatcg atagcgataa
     4981 ggatccgcgt atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc
     5041 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
     5101 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     5161 caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca
     5221 tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc
     5281 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     5341 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     5401 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     5461 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     5521 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     5581 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac
     5641 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     5701 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     5761 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     5821 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     5881 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc
     5941 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     6001 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     6061 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc
     6121 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     6181 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     6241 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     6301 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     6361 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttc
//
