LOCUS       sequence-431997-        7884 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Yeast/E. coli shuttle vector in which an RNA polymerase III promoter
            directs transcription of an RNA containing two tandem MS2 sites..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7884)
  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 7884)
  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/MS2-1
FEATURES             Location/Qualifiers
     source          1..7884
                     /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"
     misc_RNA        complement(3726..3744)
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        complement(3765..3783)
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     promoter        complement(3822..4228)
                     /gene="RPR1"
                     /label=RPR1 promoter
                     /note="promoter for the S. cerevisiae RNase P RNA gene"
     primer_bind     complement(4230..4246)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4283..4303)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4283..4301)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4322..4338)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4322..4338)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4335..4357)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4346..4362
                     /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(4370..4376,4377..4394,4395..4400))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(4553..4570)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4724..5312)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4804..4823)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5483..6274,6275..6343))
                     /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     6106..6125
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6344..6448)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      6475..7816
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(7438..7485)
                     /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     7857..7875
                     /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 gcgcgctcgt tcagaatgac acgtatagaa
     2821 tgatgcatta ccttgtcatc ttcagtatca tactgttcgt atacatactt actgacattc
     2881 ataggtatac atatatacac atgtatatat atcgtatgct gcagctttaa ataatcggtg
     2941 tcactacata agaacacctt tggtggaggg aacatcgttg gtaccattgg gcgaggtggc
     3001 ttctcttatg gcaaccgcaa gagccttgaa cgcactctca ctacggtgat gatcattctt
     3061 gcctcgcaga caatcaacgt ggagggtaat tctgctagcc tctgcaaagc ttcatggcct
     3121 ttataaaaag gaactatcca atacctcgcc agaaccaagt aacagtattt tacggggcac
     3181 aaatcaagaa caataagaca ggactgtaaa gatggacgca ttgaactccg aaagaacaac
     3241 aagagttcca aaaagtagtg gaacaaaagc aaatgaggga tttcatgcgt ttgtactcta
     3301 atctggtaga aagatgtttc acagactgtg tcaatgactt cacaacatca aagctaacca
     3361 ataaggaaca aacatgcatc atgaagtgct cagaaaagtt cttgaagcat agcgaacgtg
     3421 tagggcagcg tttccaagaa caaaacgctg ccttgggaca aggcttgggc cgataaggtg
     3481 tactggcgta tatatatcta attatgtatc tctggtgtag cccattttta gcatgtaaat
     3541 ataaagagaa accatatcta atctaaccaa atccaaacaa aattcaatag ttactatcgc
     3601 ttttttcttt ctgtatcgca aataagtgaa aattaaaaaa gaaagattaa attggaagtt
     3661 gatatggggg aattccggct agaactagtg gatcccccgg gcgatcctct agagtcgacc
     3721 tgcagacatg ggtgatcctc atgttttcta gagtcgacct gcagacatgg gtgatcctca
     3781 tgttttctag agtcgacctg caggcatgca agctggaatt ctgccaatcg cagctcccag
     3841 agtttcgttc ccagccgcga gcaccacagc gtaccatgtg cgccacgagg cctcaaacgt
     3901 aaaacaatcg aaacgaaaag aaacagacta taggggagta tagagacagc cggccaataa
     3961 gaagaggaaa aagaaatact agcgtttatc aatgtgggtc gttaacatat cctgttgaca
     4021 atgattacag gttaaaaggt agcgtaagtg aatattaact atggatattc ttatacttag
     4081 ataagagaca tataaaacac acgatgattg attgattttt atgaccaata tatgtaattc
     4141 gtaattcaga tagtttttat acttttaatg tgtgccgaac catattacta gtatatgtaa
     4201 ctaccatgtt atgttcaatt ggcagatcgg atccactagt tctagagcgg ccgccaccgc
     4261 ggtggagctc cagcttttgt tccctttagt gagggttaat tgcgcgcttg gcgtaatcat
     4321 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacataggag
     4381 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gaggtaactc acattaattg
     4441 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     4501 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     4561 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     4621 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     4681 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     4741 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     4801 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     4861 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     4921 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     4981 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     5041 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     5101 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     5161 gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     5221 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     5281 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     5341 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     5401 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     5461 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     5521 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     5581 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     5641 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     5701 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     5761 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     5821 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     5881 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     5941 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     6001 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     6061 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     6121 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     6181 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     6241 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     6301 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     6361 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     6421 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgaacgaa
     6481 gcatctgtgc ttcattttgt agaacaaaaa tgcaacgcga gagcgctaat ttttcaaaca
     6541 aagaatctga gctgcatttt tacagaacag aaatgcaacg cgaaagcgct attttaccaa
     6601 cgaagaatct gtgcttcatt tttgtaaaac aaaaatgcaa cgcgagagcg ctaatttttc
     6661 aaacaaagaa tctgagctgc atttttacag aacagaaatg caacgcgaga gcgctatttt
     6721 accaacaaag aatctatact tcttttttgt tctacaaaaa tgcatcccga gagcgctatt
     6781 tttctaacaa agcatcttag attacttttt ttctcctttg tgcgctctat aatgcagtct
     6841 cttgataact ttttgcactg taggtccgtt aaggttagaa gaaggctact ttggtgtcta
     6901 ttttctcttc cataaaaaaa gcctgactcc acttcccgcg tttactgatt actagcgaag
     6961 ctgcgggtgc attttttcaa gataaaggca tccccgatta tattctatac cgatgtggat
     7021 tgcgcatact ttgtgaacag aaagtgatag cgttgatgat tcttcattgg tcagaaaatt
     7081 atgaacggtt tcttctattt tgtctctata tactacgtat aggaaatgtt tacattttcg
     7141 tattgttttc gattcactct atgaatagtt cttactacaa tttttttgtc taaagagtaa
     7201 tactagagat aaacataaaa aatgtagagg tcgagtttag atgcaagttc aaggagcgaa
     7261 aggtggatgg gtaggttata tagggatata gcacagagat atatagcaaa gagatacttt
     7321 tgagcaatgt ttgtggaagc ggtattcgca atattttagt agctcgttac agtccggtgc
     7381 gtttttggtt ttttgaaagt gcgtcttcag agcgcttttg gttttcaaaa gcgctctgaa
     7441 gttcctatac tttctagaga ataggaactt cggaatagga acttcaaagc gtttccgaaa
     7501 acgagcgctt ccgaaaatgc aacgcgagct gcgcacatac agctcactgt tcacgtcgca
     7561 cctatatctg cgtgttgcct gtatatatat atacatgaga agaacggcat agtgcgtgtt
     7621 tatgcttaaa tgcgtactta tatgctctat ttatgtagga tgaaaggtag tctagtacct
     7681 cctgtgatat tatcccattc catgcggggt atcgtatgct tccttcagca ctacccttta
     7741 gctgttctat atgctgccac tcctcaattg gattagtctc atccttcaat gctatcattt
     7801 cctttgatat tggatcatac taagaaacca ttattatcat gacattaacc tataaaaata
     7861 ggcgtatcac gaggcccttt cgtc
//
