LOCUS       sequence-390643-        6674 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Part of Renilla reporter system, encodes Renilla luciferase with MS2
            stem loops in the 3'UTR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6674)
  AUTHORS   Luo EC, Nathanson JL, Tan FE, Schwartz JL, Schmok JC, Shankar A, 
            Markmiller S, Yee BA, Sathe S, Pratt GA, Scaletta DB, Ha Y, Hill DE,
            Aigner S, Yeo GW
  TITLE     Large-scale tethered function assays identify factors that regulate 
            mRNA stability and translation.
  JOURNAL   Nat Struct Mol Biol. 2020 Oct;27(10):989-1000. doi: 
            10.1038/s41594-020-0477-6. Epub 2020 Aug 17.
  PUBMED    32807991
REFERENCE   2  (bases 1 to 6674)
  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"; doi: 
            "10.1038/s41594-020-0477-6"; journalName: "Nat Struct Mol Biol"; 
            date: "2020-10"; volume: "27"; issue: "10"; pages: "989-1000"
COMMENT     Sequence Label: pTET2-RlucCP-3MS2
FEATURES             Location/Qualifiers
     source          1..6674
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     236..256
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        238..256
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     protein_bind    join(298..316,317..339,340..358,359..381,382..400,401..423,
                     424..442,443..465,466..484,485..507,508..526,527..549,
                     550..568)
                     /label=tetracycline response element
                     /bound_moiety="tetracycline repressor TetR"
                     /note="contains seven copies of the tetracycline operator 
                     tetO"
     promoter        601..639
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     636..660
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      691..700
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             697..1629
                     /codon_start=1
                     /product="Renilla luciferase"
                     /label=hRluc
                     /note="human codon-optimized"
                     /translation="MASKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAEN
                     AVIFLHGNAASSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAW
                     FELLNLPKKIIFVGHDWGACLAFHYSYEHQDKIKAIVHAESVVDVIESWDEWPDIEEDI
                     ALIKSEEGEKMVLENNFFVETMLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPRE
                     IPLVKGGKPDVVQIVRNYNAYLRASDDLPKMFIESDPGFFSNAIVEGAKKFPNTEFVKV
                     KGLHFSQEDAPDEMGKYIKSFVERVLKNEQ"
     CDS             1636..1683
                     /codon_start=1
                     /product="non-ORF yeast peptide conferring 
                     ubiquitin-dependent degradation"
                     /label=hCL1
                     /note="human codon-optimized"
                     /translation="ACKNWFSSLSHFVIHL"
     CDS             1687..1806
                     /codon_start=1
                     /product="PEST degradation sequence from mouse ornithine 
                     decarboxylase"
                     /label=hPEST
                     /note="human codon-optimized"
                     /translation="SHGFPPEVEEQAAGTLPMSCAQESGMDRHPAACASARINV"
     misc_RNA        1992..2010
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        2031..2049
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        2070..2088
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        2109..2127
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        2148..2166
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        2187..2205
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     promoter        complement(2227..2245)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2228..2245)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2265..2282)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2271..2495
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2541..2969
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2628..2647)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2838..2859
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2978..2998)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2983..3312
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3163..3298
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3225..3244
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3379..4173
                     /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(3433..3452)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4043..4062
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4347..4468
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4384..4403)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4438..4457
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4517..4533)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4517..4533)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4530..4552)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4541..4557
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(4565..4571,4572..4589,4590..4595))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4610..4631
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4748..4765)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4919..5507)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4999..5018)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5678..6469,6470..6538))
                     /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     6301..6320
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6539..6643)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgtgcgcaat
      241 taaccctcac taaagggaac aaaagctggg taccgggccc cccctcgagt ttaccactcc
      301 ctatcagtga tagagaaaag tgaaagtcga gtttaccact ccctatcagt gatagagaaa
      361 agtgaaagtc gagtttacca ctccctatca gtgatagaga aaagtgaaag tcgagtttac
      421 cactccctat cagtgataga gaaaagtgaa agtcgagttt accactccct atcagtgata
      481 gagaaaagtg aaagtcgagt ttaccactcc ctatcagtga tagagaaaag tgaaagtcga
      541 gtttaccact ccctatcagt gatagagaaa agtgaaagtc gagctcggta cccgggtcga
      601 ggtaggcgtg tacggtggga ggcctatata agcagagctc gtttagtgaa ccgtcagatc
      661 gcctggagac gccatccacg ctgaagctta gccaccatgg cttccaaggt gtacgacccc
      721 gagcaacgca aacgcatgat cactgggcct cagtggtggg ctcgctgcaa gcaaatgaac
      781 gtgctggact ccttcatcaa ctactatgat tccgagaagc acgccgagaa cgccgtgatt
      841 tttctgcatg gtaacgctgc ctccagctac ctgtggaggc acgtcgtgcc tcacatcgag
      901 cccgtggcta gatgcatcat ccctgatctg atcggaatgg gtaagtccgg caagagcggg
      961 aatggctcat atcgcctcct ggatcactac aagtacctca ccgcttggtt cgagctgctg
     1021 aaccttccaa agaaaatcat ctttgtgggc cacgactggg gggcttgtct ggcctttcac
     1081 tactcctacg agcaccaaga caagatcaag gccatcgtcc atgctgagag tgtcgtggac
     1141 gtgatcgagt cctgggacga gtggcctgac atcgaggagg atatcgccct gatcaagagc
     1201 gaagagggcg agaaaatggt gcttgagaat aacttcttcg tcgagaccat gctcccaagc
     1261 aagatcatgc ggaaactgga gcctgaggag ttcgctgcct acctggagcc attcaaggag
     1321 aagggcgagg ttagacggcc taccctctcc tggcctcgcg agatccctct cgttaaggga
     1381 ggcaagcccg acgtcgtcca gattgtccgc aactacaacg cctaccttcg ggccagcgac
     1441 gatctgccta agatgttcat cgagtccgac cctgggttct tttccaacgc tattgtcgag
     1501 ggagctaaga agttccctaa caccgagttc gtgaaggtga agggcctcca cttcagccag
     1561 gaggacgctc cagatgaaat gggtaagtac atcaagagct tcgtggagcg cgtgctgaag
     1621 aacgagcaga attctgcttg caagaactgg ttcagtagct taagccactt tgtgatccac
     1681 cttaacagcc acggcttccc tcccgaggtg gaggagcagg ccgccggcac cctgcccatg
     1741 agctgcgccc aggagagcgg catggataga caccctgctg cttgcgccag cgccaggatc
     1801 aacgtctaat agtgagaatt caccccacca gtgcaggctg cctatcagaa agtggtggct
     1861 ggtgtggcta atgccctggc ccacaagtat cactacgcgg ccgctcgctt tcttgctgtc
     1921 caatttctat taaaggttcc tttgttccct aagtccaact accaaactgg gtctagctct
     1981 agctgtagaa aacatgagga tcacccatgt ctgctggacg actgtagaaa acatgaggat
     2041 cacccatgtc tgctgtctag ctgtagaaaa catgaggatc acccatgtct gctggacgac
     2101 tgtagaaaac atgaggatca cccatgtctg ctgtctagct gtagaaaaca tgaggatcac
     2161 ccatgtctgc tggacgactg tagaaaacat gaggatcacc catgtctgct gtctagaggg
     2221 ccctattcta tagtgtcacc taaatgctag agctcgctga tcagcctcga ctgtgccttc
     2281 tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc
     2341 cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg
     2401 tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa
     2461 tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg
     2521 gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     2581 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
     2641 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     2701 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg
     2761 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     2821 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
     2881 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
     2941 gctgatttaa caaaaattta acgcgaatta attctgtgga atgtgtgtca gttagggtgt
     3001 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     3061 gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     3121 ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg
     3181 cccagttccg cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc
     3241 gaggccgcct ctgcctctga gctattccag aagtagtgag gaggcttttt tggaggccta
     3301 ggcttttgca aaaagctccc gggagcttgt atatccattt tcggatctga tcaagagaca
     3361 ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct
     3421 tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc
     3481 gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc
     3541 ggtgccctga atgaactgca ggacgaggca gcgcggctat cgtggctggc cacgacgggc
     3601 gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg
     3661 ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc
     3721 atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac
     3781 caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat
     3841 caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc
     3901 aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg
     3961 aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg
     4021 gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc
     4081 gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc
     4141 gccttctatc gccttcttga cgagttcttc tgagcgggac tctggggttc gaaatgaccg
     4201 accaagcgac gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa
     4261 ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc
     4321 tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat
     4381 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     4441 gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga
     4501 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     4561 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     4621 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     4681 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     4741 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     4801 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     4861 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     4921 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     4981 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     5041 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     5101 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     5161 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     5221 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     5281 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     5341 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     5401 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     5461 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     5521 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     5581 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     5641 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     5701 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     5761 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     5821 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     5881 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     5941 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     6001 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     6061 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     6121 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     6181 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     6241 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     6301 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     6361 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     6421 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     6481 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     6541 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     6601 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     6661 tgccacctga cgtc
//
