LOCUS       sequence-437365-        7879 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For expressing an RNA containing your sequence of interest adjacent 
            to two MS2 sites from an RNA polymerase III promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7879)
  AUTHORS   Bernstein DS, Buter N, Stumpf C, Wickens M
  TITLE     Analyzing mRNA-protein complexes using a yeast three-hybrid system.
  JOURNAL   Methods. 2002 Feb;26(2):123-41. doi: 10.1016/S1046-2023(02)00015-4.
  PUBMED    12054889
REFERENCE   2  (bases 1 to 7879)
  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"; journalName: 
            "Methods"; date: "2002-02"; volume: "26"; issue: "2"; pages: 
            "123-41"
COMMENT     Sequence Label: pIII/MS2-2
FEATURES             Location/Qualifiers
     source          1..7879
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        194..409
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             410..1213
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     rep_origin      complement(1344..1799)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1481..1502)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1693..1712
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1925..1947
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1939..1956
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1940..1956
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1966..1985
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1966..1984
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     2213..2232
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     terminator      complement(3595..3657)
                     /label=RPR1 terminator
                     /note="transcription terminator for the S. cerevisiae RNase
                     P RNA gene"
     misc_RNA        complement(3690..3708)
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        complement(3729..3747)
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     promoter        complement(3812..4218)
                     /gene="RPR1"
                     /label=RPR1 promoter
                     /note="promoter for the S. cerevisiae RNase P RNA gene"
     primer_bind     complement(4224..4240)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4277..4297)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4277..4295)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4316..4332)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4316..4332)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4329..4351)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4340..4356
                     /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(4364..4370,4371..4388,4389..4394))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4409..4430
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4547..4564)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4718..5306)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4798..4817)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5477..6268,6269..6337))
                     /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     6100..6119
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6338..6442)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      6469..7811
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(7432..7479)
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     primer_bind     7852..7870
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accataccac cttttcaatt catcattttt tttttattct tttttttgat ttcggtttcc
      241 ttgaaatttt tttgattcgg taatctccga acagaaggaa gaacgaagga aggagcacag
      301 acttagattg gtatatatac gcatatgtag tgttgaagaa acatgaaatt gcccagtatt
      361 cttaacccaa ctgcacagaa caaaaacctg caggaaacga agataaatca tgtcgaaagc
      421 tacatataag gaacgtgctg ctactcatcc tagtcctgtt gctgccaagc tatttaatat
      481 catgcacgaa aagcaaacaa acttgtgtgc ttcattggat gttcgtacca ccaaggaatt
      541 actggagtta gttgaagcat taggtcccaa aatttgttta ctaaaaacac atgtggatat
      601 cttgactgat ttttccatgg agggcacagt taagccgcta aaggcattat ccgccaagta
      661 caatttttta ctcttcgaag acagaaaatt tgctgacatt ggtaatacag tcaaattgca
      721 gtactctgcg ggtgtataca gaatagcaga atgggcagac attacgaatg cacacggtgt
      781 ggtgggccca ggtattgtta gcggtttgaa gcaggcggca gaagaagtaa caaaggaacc
      841 tagaggcctt ttgatgttag cagaattgtc atgcaagggc tccctatcta ctggagaata
      901 tactaagggt actgttgaca ttgcgaagag cgacaaagat tttgttatcg gctttattgc
      961 tcaaagagac atgggtggaa gagatgaagg ttacgattgg ttgattatga cacccggtgt
     1021 gggtttagat gacaagggag acgcattggg tcaacagtat agaaccgtgg atgatgtggt
     1081 ctctacagga tctgacatta ttattgttgg aagaggacta tttgcaaagg gaagggatgc
     1141 taaggtagag ggtgaacgtt acagaaaagc aggctgggaa gcatatttga gaagatgcgg
     1201 ccagcaaaac taaaaaactg tattataagt aaatgcatgt atactaaact cacaaattag
     1261 agcttcaatt taattatatc agttattacc ctatgcggtg tgaaataccg cacagatgcg
     1321 taaggagaaa ataccgcatc aggaaattgt aagcgttaat attttgttaa aattcgcgtt
     1381 aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta
     1441 taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc
     1501 actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg
     1561 cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact
     1621 aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt
     1681 ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc
     1741 ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc
     1801 cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta
     1861 ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg
     1921 ttttcccagt cacgacgttg taaaacgacg gccagtgagc gcgcgtaata cgactcacta
     1981 tagggcgaat tgggtaccgg gccccccctc gaggtcgacg ctctccctta tgcgactcct
     2041 gcattaggaa gcagcccagt agtaggttga ggccgttgag caccgccgcc gcaaggaatg
     2101 gtgcatgcaa ggagatggcg cccaacagtc ccccggccac ggggcctgcc accataccca
     2161 cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt
     2221 cggcgatata ggcgccagca accgcacctg tggcgccggt gatgccggcc acgatgcgtc
     2281 cggcgtagag gatcgatcct cggggacacc aaatatggcg atctcggcct tttcgtttct
     2341 tggagctggg acatgtttgc catcgatcca tctaccacca gaacggccgt tagatctgct
     2401 gccaccgttg tttccaccga agaaaccacc gttgccgtaa ccaccacgac ggttgttgct
     2461 aaagaagctg ccaccgccac ggccaccgtt gtagccgccg ttgttgttat tgtagttgct
     2521 actgttattt ctggcacttc ttggttttcc tcttaagtga ggaggaacat aaccattctc
     2581 gttgttgtcg ttgatgctta aattttgcac ttgttcgctc agttcagcca taatatgaaa
     2641 tgcttttctt gttgttctta cggaatacca cttgccacct atcaccacaa ctaacttttt
     2701 cccgttcctc catctctttt atattttttt tctcgagttc aagagaaaaa aaaagaaaaa
     2761 gcaaaaagaa aaaaggaaag cgcgcctcgt tcagaatgac acgtatagaa tgatgcatta
     2821 ccttgtcatc ttcagtatca tactgttcgt atacatactt actgacattc ataggtatac
     2881 atatatacac atgtatatat atcgtatgct gcagctttaa ataatcggtg tcactacata
     2941 agaacacctt tggtggaggg aacatcgttg gtaccattgg gcgaggtggc ttctcttatg
     3001 gcaaccgcaa gagccttgaa cgcactctca ctacggtgat gatcattctt gcctcgcaga
     3061 caatcaacgt ggagggtaat tctgctagcc tctgcaaagc ttcatggcct ttataaaaag
     3121 gaactatcca atacctcgcc agaaccaagt aacagtattt tacggggcac aaatcaagaa
     3181 caataagaca ggactgtaaa gatggacgca ttgaactcca aagaacaaca agagttccaa
     3241 aaagtagtgg aacaaaagca aatgaaggat ttcatgcgtt tgtactctaa tctggtagaa
     3301 agatgtttca cagactgtgt caatgacttc acaacatcaa agctaaccaa taaggaacaa
     3361 acatgcatca tgaagtgctc agaaaagttc ttgaagcata gcgaacgtgt agggcagcgt
     3421 ttccaagaac aaaacgctgc cttgggacaa ggcttgggcc gataaggtgt actggcgtat
     3481 atatatctaa ttatgtatct ctggtgtagc ccatttttag catgtaaata taaagagaaa
     3541 ccatatctaa tctaaccaaa tccaaacaaa attcaatagt tactatcgct tttttctttc
     3601 tgtatcgcaa ataagtgaaa attaaaaaag aaagattaaa ttggaagttg gatatggggg
     3661 aattccgatc ctctagagtc gacctgcaga catgggtgat cctcatgttt tctagagtcg
     3721 acctgcagac atgggtgatc ctcatgtttt ctagagtcga cctgcaggca tgcaagctgc
     3781 ccgggggatc cactagttct agccggaatt ctgccaatcg cagctcccag agtttcgttc
     3841 ccagccgcga gcaccacagc gtaccatgtg cgccacgagg cctcaaacgt aaaacaatcg
     3901 aaacgaaaag aaacagacta taggggagta tagagacagc cggccaataa gaagaggaaa
     3961 aagaaatact agcgtttatc aatgtgggtc gttaacatat cctgttgaca atgattacag
     4021 gttaaaaggt agcgtaagtg aatattaact atggatattc ttatacttag ataagagaca
     4081 tataaaacac acgatgattg attgattttt atgaccaata tatgtaattc gtaattcaga
     4141 tagtttttat acttttaatg tgtgccgaac catattacta gtatatgtaa ctaccatgtt
     4201 atgttcaatt ggcagatccc ccggatccac tagttctaga gcggccgcca ccgcggtgga
     4261 gctccagctt ttgttccctt tagtgagggt taattgcgcg cttggcgtaa tcatggtcat
     4321 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     4381 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     4441 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     4501 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     4561 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     4621 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     4681 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     4741 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     4801 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     4861 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     4921 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     4981 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     5041 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     5101 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     5161 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     5221 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     5281 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     5341 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     5401 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     5461 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     5521 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     5581 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     5641 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     5701 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     5761 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     5821 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     5881 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     5941 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     6001 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     6061 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     6121 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     6181 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     6241 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     6301 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     6361 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     6421 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgaa cgaagcatct
     6481 gtgcttcatt ttgtagaaca aaaatgcaac gcgagagcgc taatttttca aacaaagaat
     6541 ctgagctgca tttttacaga acagaaatgc aacgcgaaag cgctatttta ccaacgaaga
     6601 atctgtgctt catttttgta aaacaaaaat gcaacgcgag agcgctaatt tttcaaacaa
     6661 agaatctgag ctgcattttt acagaacaga aatgcaacgc gagagcgcta ttttaccaac
     6721 aaagaatcta tacttctttt ttgttctaca aaaatgcatc ccgagagcgc tatttttcta
     6781 acaaagcatc ttagattact ttttttctcc tttgtgcgct ctataatgca gtctcttgat
     6841 aactttttgc actgtaggtc cgttaaggtt agaagaaggc tactttggtg tctattttct
     6901 cttccataaa aaaagcctga ctccacttcc cgcgtttact gattactagc gaagctgcgg
     6961 gtgcattttt tcaagataaa ggcatccccg attatattct ataccgatgt ggattgcgca
     7021 tactttgtga acagaaagtg atagcgttga tgattcttca ttggtcagaa aattatgaac
     7081 ggtttcttct attttgtctc tatatactac gtataggaaa tgtttacatt ttcgtattgt
     7141 tttcgattca ctctatgaat agttcttact acaatttttt tgtctaaaga gtaatactag
     7201 agataaacat aaaaaatgta gaggtcgagt ttagatgcaa gttcaaggag cgaaaggtgg
     7261 atgggtaggt tatataggga tatagcacag agatatatag caaagagata cttttgagca
     7321 atgtttgtgg aagcggtatt cgcaatattt tagtagctcg ttacagtccg gtgcgttttt
     7381 ggttttttga aagtgcgtct tcagagcgct tttggttttc aaaagcgctc tgaagttcct
     7441 atactttcta gagaatagga acttcggaat aggaacttca aagcgtttcc gaaaacgagc
     7501 gcttccgaaa atgcaacgcg agctgcgcac atacagctca ctgttcacgt cgcacctata
     7561 tctgcgtgtt gcctgtatat atatatacat gagaagaacg gcatagtgcg tgtttatgct
     7621 taaatgcgta cttatatgcg tctatttatg taggatgaaa ggtagtctag tacctcctgt
     7681 gatattatcc cattccatgc ggggtatcgt atgcttcctt cagcactacc ctttagctgt
     7741 tctatatgct gccactcctc aattggatta gtctcatcct tcaatgctat catttccttt
     7801 gatattggat catactaaga aaccattatt atcatgacat taacctataa aaataggcgt
     7861 atcacgaggc cctttcgtc
//
