LOCUS       sequence-436938-        7084 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cassette vector to enable overexpression of rNTases as MS2 coat 
            protein fusions in S. cerevisiae..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7084)
  AUTHORS   Preston MA, Porter DF, Chen F, Buter N, Lapointe CP, Keles S, Kimble
            J, Wickens M
  TITLE     Unbiased screen of RNA tailing activities reveals a poly(UG) 
            polymerase.
  JOURNAL   Nat Methods. 2019 May;16(5):437-445. doi: 10.1038/s41592-019-0370-6.
            Epub 2019 Apr 15.
  PUBMED    30988468
REFERENCE   2  (bases 1 to 7084)
  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/s41592-019-0370-6"; journalName: "Nat Methods"; date: 
            "2019-05"; volume: "16"; issue: "5"; pages: "437-445"
COMMENT     Sequence Label: MAP72_MS2
FEATURES             Location/Qualifiers
     source          1..7084
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(436..452)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(436..452)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(449..471)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    460..476
                     /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(484..490,491..508,509..514))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    529..550
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(667..684)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(838..1426)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(918..937)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1597..2388,2389..2457))
                     /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     2220..2239
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2458..2562)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2630..2648
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2668..3073
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             3074..4168
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKADAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     rep_origin      4306..5652
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     primer_bind     6074..6096
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6088..6105
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6089..6105
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     terminator      complement(6113..6299)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     6220..6244
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     CDS             complement(6437..6454)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(6464..6484)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(6527..6913)
                     /codon_start=1
                     /product="bacteriophage MS2 coat protein"
                     /label=MS2-N55K
                     /note="binds with high affinity to a specific stem-loop 
                     structure in the viral RNA (Lim et al., 1994)"
                     /translation="ASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVT
                     CSVRQSSAQKRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELI
                     VKAMQGLLKDGNPIPSAIAANSGIY"
     CDS             complement(6527..6913)
                     /codon_start=1
                     /product="bacteriophage MS2 coat protein"
                     /label=MS2
                     /note="binds to a specific stem-loop structure in the viral
                     RNA (Peabody, 1993)"
                     /translation="ASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVT
                     CSVRQSSAQKRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELI
                     VKAMQGLLKDGNPIPSAIAANSGIY"
     promoter        complement(join(6936..7084,1..265))
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
ORIGIN
        1 aaaaaaaaaa attttcaaga aaaagaaacg tgataaaaat ttttattgcc tttttcgacg
       61 aagaaaaaga aacgaggcgg tctctttttt cttttccaaa cctttagtac gggtaattaa
      121 cgacacccta gaggaagaaa gaggggaaat ttagtatgct gtgcttgggt gttttgaagt
      181 ggtacggcga tgcgcggagt ccgagaaaat ctggaagagt aaaaaaggag tagaaacatt
      241 ttgaagctat ggtgtgtggg ggatcacttg tgggggattg ggtgtgatgt aaggattcgc
      301 ggtcctcgaa aattaaaagt ccaacgcgcc tgttgcttcc tatgtgatat gtattatatg
      361 taatatgcat aaatatatct actgcattgt attttgaacg tacaaagtat gcattgtaag
      421 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
      481 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
      541 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
      601 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
      661 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
      721 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
      781 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
      841 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
      901 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
      961 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     1021 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     1081 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     1141 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     1201 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     1261 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     1321 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     1381 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     1441 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     1501 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     1561 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     1621 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     1681 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     1741 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     1801 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     1861 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     1921 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     1981 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     2041 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     2101 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     2161 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     2221 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     2281 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     2341 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     2401 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     2461 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     2521 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     2581 gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat
     2641 cacgaggccc tttcgtcttc aagaattaac tgtgggaata ctcaggtatc gtaagatgca
     2701 agagttcgaa tctcttagca accattattt ttttcctcaa cataacgaga acacacaggg
     2761 gcgctatcgc acagaatcaa attcgatgac tggaaatttt ttgttaattt cagaggtcgc
     2821 ctgacgcata tacctttttc aactgaaaaa ttgggagaaa aaggaaaggt gagagcgccg
     2881 gaaccggctt ttcatataga atagagaagc gttcatgact aaatgcttgc atcacaatac
     2941 ttgaagttga caatattatt taaggaccta ttgttttttc caataggtgg ttagcaatcg
     3001 tcttactttc taacttttct taccttttac atttcagcaa tatatatata tatttcaagg
     3061 atataccatt ctaatgtctg cccctaagaa gatcgtcgtt ttgccaggtg accacgttgg
     3121 tcaagaaatc acagccgaag ccattaaggt tcttaaagct atttctgatg ttcgttccaa
     3181 tgtcaagttc gatttcgaaa atcatttaat tggtggtgct gctatcgatg ctacaggtgt
     3241 tccacttcca gatgaggcgc tggaagcctc caagaaggct gatgccgttt tgttaggtgc
     3301 tgtgggtggt cctaaatggg gtacaggtag tgttagacct gaacaaggtt tactaaaaat
     3361 ccgtaaagaa cttcaattgt acgccaactt aagaccatgt aactttgcat ccgactctct
     3421 tttagactta tctccaatca agccacaatt tgctaaaggt actgacttcg ttgttgtcag
     3481 agaattagtg ggaggtattt actttggtaa gagaaaggaa gacgatggtg atggtgtcgc
     3541 ttgggatagt gaacaataca ccgttccaga agtgcaaaga atcacaagaa tggccgcttt
     3601 catggcccta caacatgagc caccattgcc tatttggtcc ttggataaag ctaatgtttt
     3661 ggcctcttca agattatgga gaaaaactgt ggaggaaacc atcaagaacg aatttcctac
     3721 attgaaggtt caacatcaat tgattgattc tgccgccatg atcctagtta agaacccaac
     3781 ccacctaaat ggtattataa tcaccagcaa catgtttggt gatatcatct ccgatgaagc
     3841 ctccgttatc ccaggttcct tgggtttgtt gccatctgcg tccttggcct ctttgccaga
     3901 caagaacacc gcatttggtt tgtacgaacc atgccacggt tctgctccag atttgccaaa
     3961 gaataaggtc aaccctatcg ccactatctt gtctgctgca atgatgttga aattgtcatt
     4021 gaacttgcct gaagaaggta aggccattga agatgcagtt aaaaaggttt tggatgcagg
     4081 tatcagaact ggtgatttag gtggttccaa cagtaccacc gaagtcggtg atgctgtcgc
     4141 cgaagaagtt aagaaaatcc ttgcttaaaa agattctctt tttttatgat atttgtacat
     4201 aaactttata aatgaaattc ataatagaaa cgacacgaaa ttacaaaatg gaatatgttc
     4261 atagggtaga attaattctc atgtttgaca gcttatcatc ggatcgatcc aatatcaaag
     4321 gaaatgatag cattgaagga tgagactaat ccaattgagg agtggcagca tatagaacag
     4381 ctaaagggta gtgctgaagg aagcatacga taccccgcat ggaatgggat aatatcacag
     4441 gaggtactag actacctttc atcctacata aatagacgca tataagtacg catttaagca
     4501 taaacacgca ctatgccgtt cttctcatgt atatatatat acaggcaaca cgcagatata
     4561 ggtgcgacgt gaacagtgag ctgtatgtgc gcagctcgcg ttgcattttc ggaagcgctc
     4621 gttttcggaa acgctttgaa gttcctattc cgaagttcct attctctagc tagaaagtat
     4681 aggaacttca gagcgctttt gaaaaccaaa agcgctctga agacgcactt tcaaaaaacc
     4741 aaaaacgcac cggactgtaa cgagctacta aaatattgcg aataccgctt ccacaaacat
     4801 tgctcaaaag tatctctttg ctatatatct ctgtgctata tccctatata acctacccat
     4861 ccacctttcg ctccttgaac ttgcatctaa actcgacctc tacatttttt atgtttatct
     4921 ctagtattac tctttagaca aaaaaattgt agtaagaact attcatagag tgaatcgaaa
     4981 acaatacgaa aatgtaaaca tttcctatac gtagtatata gagacaaaat agaagaaacc
     5041 gttcataatt ttctgaccaa tgaagaatca tcaacgctat cactttctgt tcacaaagta
     5101 tgcgcaatcc acatcggtat agaatataat cggggatgcc tttatcttga aaaaatgcac
     5161 ccgcagcttc gctagtaatc agtaaacgcg ggaagtggag tcaggctttt tttatggaag
     5221 agaaaataga caccaaagta gccttcttct aaccttaacg gacctacagt gcaaaaagtt
     5281 atcaagagac tgcattatag agcgcacaaa ggagaaaaaa agtaatctaa gatgctttgt
     5341 tagaaaaata gcgctctcgg gatgcatttt tgtagaacaa aaaagaagta tagattcttt
     5401 gttggtaaaa tagcgctctc gcgttgcatt tctgttctgt aaaaatgcag ctcagattct
     5461 ttgtttgaaa aattagcgct ctcgcgttgc atttttgttt tacaaaaatg aagcacagat
     5521 tcttcgttgg taaaatagcg ctttcgcgtt gcatttctgt tctgtaaaaa tgcagctcag
     5581 attctttgtt tgaaaaatta gcgctctcgc gttgcatttt tgttctacaa aatgaagcac
     5641 agatgcttcg ttaacaaaga tatgctattg aagtgcaaga tggaaacgca gaaaatgaac
     5701 cggggatgcg acgtgcaaga ttacctatgc aatagatgca atagtttctc caggaaccga
     5761 aatacataca ttgtcttccg taaagcgcta gactatatat tattatacag gttcaaatat
     5821 actatctgtt tcagggaaaa ctcccaggtt cggatgttca aaattcaatg atgggtaaca
     5881 agtacgatcc gataaatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc
     5941 atcaggcgcc attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc
     6001 tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta
     6061 acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgctcg agcggtagag
     6121 gtgtggtcaa taagagcgac ctcatgctat acctgagaaa gcaacctgac ctacaggaaa
     6181 gagttactca agaataagaa ttttcgtttt aaaacctaag agtcacttta aaatttgtat
     6241 acacttattt tttttataac ttatttaata ataaaaatca taaatcataa gaaattcgct
     6301 tatttagaag tgtcaacaac gtatctacca acgatttgac ccttttccat cttttcgtaa
     6361 atttctggca aggtagacaa gccgacaacc ttgattggag acttgaccaa acctctggcg
     6421 aagaagtcca aagttagtgg tggtggtggt ggtgagaacc tctaaccttt ctcttcttct
     6481 ttggggtacc ctgcagtcta gaaggcggcc gcccggggga tccgacgtag atgccggagt
     6541 ttgctgcgat ggccgaggga atcgggtttc catcttttag gagaccttgc attgccttaa
     6601 caataagctc gcagtcggaa ttcgtggcga aaattggaat ggttagttcc atatttaagt
     6661 acgaacgcca tgcggctaca ggaagctcta caccaccaac agtctgggtt gccactttag
     6721 gcacctcgac tttgatggtg tatttgcgct tctgcgcaga gctctgacga acgctacagg
     6781 ttactttgta agcctgtgaa cgcgagttag agctgatcca ttcagcgacc ccgttagcga
     6841 agttgcttgg ggcgacagtc acgtcgccag ttccgccatt gtcgacgaga acgaactgag
     6901 taaagttaga agccatacta gttttgtaat taaaacttag attagattgc tatgctttct
     6961 ttctaatgag caagaagtaa aaaaagttgt aatagaacaa gaaaaatgaa actgaaactt
     7021 gagaaattga agaccgttta ttaacttaaa tatcaatggg aggtcatcga aagagaaaaa
     7081 aatc
//
