LOCUS       sequence-432002-        7887 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 7887)
  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 7887)
  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..7887
                     /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        196..416
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             417..1220
                     /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(1351..1806)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1488..1509)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1700..1719
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1936..1958
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1950..1967
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1951..1967
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1977..1996
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1977..1995
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     2224..2243
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     terminator      complement(3607..3669)
                     /label=RPR1 terminator
                     /note="transcription terminator for the S. cerevisiae RNase
                     P RNA gene"
     misc_RNA        complement(3702..3720)
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        complement(3741..3759)
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     promoter        complement(3824..4230)
                     /gene="RPR1"
                     /label=RPR1 promoter
                     /note="promoter for the S. cerevisiae RNase P RNA gene"
     primer_bind     complement(4232..4248)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4285..4305)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4285..4303)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4324..4340)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4324..4340)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4337..4359)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4348..4364
                     /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(4372..4378,4379..4396,4397..4402))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(4555..4572)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4726..5314)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4806..4825)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5485..6276,6277..6345))
                     /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     6108..6127
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6346..6450)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      6477..7819
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(7440..7487)
                     /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     7860..7878
                     /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 agcttttcaa ttcaattcat catttttttt ttattctttt ttttgatttc
      241 ggtttctttg aaattttttt gattcggtaa tctccgaaca gaaggaagaa cgaaggaagg
      301 agcacagact tagattggta tatatacgca tatgtagtgt tgaagaaaca tgaaattgcc
      361 cagtattctt aacccaactg cacagaacaa aaacctgcag gaaacgaaga taaatcatgt
      421 cgaaagctac atataaggaa cgtgctgcta ctcatcctag tcctgttgct gccaagctat
      481 ttaatatcat gcacgaaaag caaacaaact tgtgtgcttc attggatgtt cgtaccacca
      541 aggaattact ggagttagtt gaagcattag gtcccaaaat ttgtttacta aaaacacatg
      601 tggatatctt gactgatttt tccatggagg gcacagttaa gccgctaaag gcattatccg
      661 ccaagtacaa ttttttactc ttcgaagaca gaaaatttgc tgacattggt aatacagtca
      721 aattgcagta ctctgcgggt gtatacagaa tagcagaatg ggcagacatt acgaatgcac
      781 acggtgtggt gggcccaggt attgttagcg gtttgaagca ggcggcagaa gaagtaacaa
      841 aggaacctag aggccttttg atgttagcag aattgtcatg caagggctcc ctatctactg
      901 gagaatatac taagggtact gttgacattg cgaagagcga caaagatttt gttatcggct
      961 ttattgctca aagagacatg ggtggaagag atgaaggtta cgattggttg attatgacac
     1021 ccggtgtggg tttagatgac aagggagacg cattgggtca acagtataga accgtggatg
     1081 atgtggtctc tacaggatct gacattatta ttgttggaag aggactattt gcaaagggaa
     1141 gggatgctaa ggtagagggt gaacgttaca gaaaagcagg ctgggaagca tatttgagaa
     1201 gatgcggcca gcaaaactaa aaaactgtat tataagtaaa tgcatgtata ctaaactcac
     1261 aaattagagc ttcaatttaa ttatatcagt tattacccta tgcggtgtga aataccgcac
     1321 agatgcgtaa ggagaaaata ccgcatcagg aaattgtaaa cgttaatatt ttgttaaaat
     1381 tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa
     1441 tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca
     1501 agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg
     1561 gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta
     1621 aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg
     1681 cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa
     1741 gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg
     1801 gcgcgtcgcg ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg
     1861 cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg
     1921 taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgag cgcgcgtaat
     1981 acgactcact atagggcgaa ttgggtaccg ggccccccct cgaggtcgac gctctccctt
     2041 atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc
     2101 cgcaaggaat ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc
     2161 caccataccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc
     2221 atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc
     2281 cacgatgcgt ccggcgtaga ggatcgatcc tcggggacac caaatatggc gatctcggcc
     2341 ttttcgtttc ttggagctgg gacatgtttg ccatcgatcc atctaccacc agaacggccg
     2401 ttagatctgc tgccaccgtt gtttccaccg aagaaaccac cgttgccgta accaccacga
     2461 cggttgttgc taaagaagct gccaccgcca cggccaccgt tgtagccgcc gttgttgtta
     2521 ttgtagttgc tactgttatt tctggcactt cttggttttc ctcttaagtg aggaggaaca
     2581 taaccattct cgttgttgtc gttgatgctt aaattttgca cttgttcgct cagttcagcc
     2641 ataatatgaa atgcttttct tgttgttctt acggaatacc acttgccacc tatcaccaca
     2701 actaactttt tcccgttcct ccatctcttt tatatttttt ttctcgagtt caagagaaaa
     2761 aaaaagaaaa agcaaaaaga aaaaaggaaa gcgcgcctcg ttcagaatga cacgtataga
     2821 atgatgcatt accttgtcat cttcagtatc atactgttcg tatacatact tactgacatt
     2881 cataggtata catatataca catgtatata tatcgtatgc tgcagcttta aataatcggt
     2941 gtcactacat aagaacacct ttggtggagg gaacatcgtt ggtaccattg ggcgaggtgg
     3001 cttctcttat ggcaaccgca agagccttga acgcactctc actacggtga tgatcattct
     3061 tgcctcgcag acaatcaacg tggagggtaa ttctgctagc ctctgcaaag cttcatggcc
     3121 tttataaaaa ggaactatcc aatacctcgc cagaaccaag taacagtatt ttacggggca
     3181 caaatcaaga acaataagac aggactgtaa agatggacgc attgaactcc gaaagaacaa
     3241 caagagttcc aaaaagtagt ggaacaaaag caaatgaggg atttcatgcg tttgtactct
     3301 aatctggtag aaagatgttt cacagactgt gtcaatgact tcacaacatc aaagctaacc
     3361 aataaggaac aaacatgcat catgaagtgc tcagaaaagt tcttgaagca tagcgaacgt
     3421 gtagggcagc gtttccaaga acaaaacgct gccttgggac aaggcttggg ccgataaggt
     3481 gtactggcgt atatatatct aattatgtat ctctggtgta gcccattttt agcatgtaaa
     3541 tataaagaga aaccatatct aatctaacca aatccaaaca aaattcaata gttactatcg
     3601 cttttttctt tctgtatcgc aaataagtga aaattaaaaa agaaagatta aattggaagt
     3661 tggatatggg ggaattccga tcctctagag tcgacctgca gacatgggtg atcctcatgt
     3721 tttctagagt cgacctgcag acatgggtga tcctcatgtt ttctagagtc gacctgcagg
     3781 catgcaagct gcccggggga tccactagtt ctagccggaa ttctgccaat cgcagctccc
     3841 agagtttcgt tcccagccgc gagcaccaca gcgtaccatg tgcgccacga ggcctcaaac
     3901 gtaaaacaat cgaaacgaaa agaaacagac tataggggag tatagagaca gccggccaat
     3961 aagaagagga aaaagaaata ctagcgttta tcaatgtggg tcgttaacat atcctgttga
     4021 caatgattac aggttaaaag gtagcgtaag tgaatattaa ctatggatat tcttatactt
     4081 agataagaga catataaaac acacgatgat tgattgattt ttatgaccaa tatatgtaat
     4141 tcgtaattca gatagttttt atacttttaa tgtgtgccga accatattac tagtatatgt
     4201 aactaccatg ttatgttcaa ttggcagatc ggatccacta gttctagagc ggccgccacc
     4261 gcggtggagc tccagctttt gttcccttta gtgagggtta attgcgcgct tggcgtaatc
     4321 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatagg
     4381 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgaggtaac tcacattaat
     4441 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     4501 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     4561 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     4621 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     4681 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     4741 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     4801 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     4861 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     4921 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     4981 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     5041 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     5101 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     5161 tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     5221 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     5281 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     5341 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     5401 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     5461 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     5521 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     5581 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     5641 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     5701 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     5761 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     5821 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     5881 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     5941 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     6001 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     6061 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     6121 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     6181 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     6241 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     6301 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     6361 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     6421 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgaacg
     6481 aagcatctgt gcttcatttt gtagaacaaa aatgcaacgc gagagcgcta atttttcaaa
     6541 caaagaatct gagctgcatt tttacagaac agaaatgcaa cgcgaaagcg ctattttacc
     6601 aacgaagaat ctgtgcttca tttttgtaaa acaaaaatgc aacgcgagag cgctaatttt
     6661 tcaaacaaag aatctgagct gcatttttac agaacagaaa tgcaacgcga gagcgctatt
     6721 ttaccaacaa agaatctata cttctttttt gttctacaaa aatgcatccc gagagcgcta
     6781 tttttctaac aaagcatctt agattacttt ttttctcctt tgtgcgctct ataatgcagt
     6841 ctcttgataa ctttttgcac tgtaggtccg ttaaggttag aagaaggcta ctttggtgtc
     6901 tattttctct tccataaaaa aagcctgact ccacttcccg cgtttactga ttactagcga
     6961 agctgcgggt gcattttttc aagataaagg catccccgat tatattctat accgatgtgg
     7021 attgcgcata ctttgtgaac agaaagtgat agcgttgatg attcttcatt ggtcagaaaa
     7081 ttatgaacgg tttcttctat tttgtctcta tatactacgt ataggaaatg tttacatttt
     7141 cgtattgttt tcgattcact ctatgaatag ttcttactac aatttttttg tctaaagagt
     7201 aatactagag ataaacataa aaaatgtaga ggtcgagttt agatgcaagt tcaaggagcg
     7261 aaaggtggat gggtaggtta tatagggata tagcacagag atatatagca aagagatact
     7321 tttgagcaat gtttgtggaa gcggtattcg caatatttta gtagctcgtt acagtccggt
     7381 gcgtttttgg ttttttgaaa gtgcgtcttc agagcgcttt tggttttcaa aagcgctctg
     7441 aagttcctat actttctaga gaataggaac ttcggaatag gaacttcaaa gcgtttccga
     7501 aaacgagcgc ttccgaaaat gcaacgcgag ctgcgcacat acagctcact gttcacgtcg
     7561 cacctatatc tgcgtgttgc ctgtatatat atatacatga gaagaacggc atagtgcgtg
     7621 tttatgctta aatgcgtact tatatgcgtc tatttatgta ggatgaaagg tagtctagta
     7681 cctcctgtga tattatccca ttccatgcgg ggtatcgtat gcttccttca gcactaccct
     7741 ttagctgttc tatatgctgc cactcctcaa ttggattagt ctcatccttc aatgctatca
     7801 tttcctttga tattggatca tactaagaaa ccattattat catgacatta acctataaaa
     7861 ataggcgtat cacgaggccc tttcgtc
//
