LOCUS       sequence-436947-        7721 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Construct used to generate the PUF3/PUP2/3HA tagging strains. (aka 
            pBSII/PUF3 5' flank/PUP2-DADA/3HA/URA3/PUF3 3' flank).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7721)
  AUTHORS   Lapointe CP, Wilinski D, Saunders HA, Wickens M
  TITLE     Protein-RNA networks revealed through covalent RNA marks.
  JOURNAL   Nat Methods. 2015 Dec;12(12):1163-70. doi: 10.1038/nmeth.3651. Epub 
            2015 Nov 2.
  PUBMED    26524240
REFERENCE   2  (bases 1 to 7721)
  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/nmeth.3651"; journalName: "Nat Methods"; date: "2015-12"; 
            volume: "12"; issue: "12"; pages: "1163-70"
COMMENT     Sequence Label: RT-5
FEATURES             Location/Qualifiers
     source          1..7721
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             3361..3450
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     CDS             complement(3554..4357)
                     /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"
     primer_bind     complement(5487..5503)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5488..5504)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(5533..5553)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5533..5551)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5572..5588)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5572..5588)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5585..5607)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5596..5612
                     /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(5620..5626,5627..5644,5645..5650))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5665..5686
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5803..5820)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5974..6562)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6054..6073)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6733..7524,7525..7593))
                     /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     7356..7375
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7594..7698)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tggagctcca
      661 ccgcggtggc ggccgcctca aagtctacag caacagcagc aaacgtatca tcgatcgccg
      721 ttgttagagc aactaagaaa cagtagttct gacaaaaatt caaactcgaa catgtcacta
      781 aaagacatct ttggccattc cttagagttt tgcaaagatc aacatggttc acggttcatt
      841 cagcgcgagt tggccacctc accagcctca gaaaaggagg tcatattcaa tgaaattcgc
      901 gatgacgcca ttgagctttc gaacgatgta tttggtaatt acgtcattca gaagtttttt
      961 gaatttggta gcaaaattca aaaaaatact ttggttgatc aatttaaagg taacatgaaa
     1021 cagttatctc tacaaatgta cgcatgtcgt gtaattcaaa aggcgttaga atatattgat
     1081 tccaatcaaa gaattgagtt agtcctggaa ttatctgact ccgttttgca aatgatcaaa
     1141 gatcagaacg gtaatcacgt aattcaaaag gccatcgaaa caattcctat cgaaaaatta
     1201 ccttttatct taagttcttt aactggccac atttaccatc tatctaccca ctcctacggc
     1261 tgtagggtca ttcaaagatt gttagaattt ggttcgagcg aagatcagga aagcatttta
     1321 aacgaattga aagatttcat accatatcta attcaagacc aatatggtaa ctatgtcatt
     1381 caatatgttt tgcaacaaga tcagttcacc aataaggaaa tggtcgatat caaacaggaa
     1441 atcatagaaa ctgtggccaa taatgttgtc gaatactcta aacataaatt tgcctccaac
     1501 gtggtcgaaa aatctattct ttatggctct aaaaatcaga aggatttaat aatttctaag
     1561 attttaccaa gagacaaaaa tcatgctttg aatctggaag acgattctcc aatgatttta
     1621 atgatcaagg atcaatttgc caactatgtg attcaaaaat tagttaatgt ctctgagggt
     1681 gagggtaaga aattgatcgt cattgccatt agagcatatt tggataaact aaacaaatct
     1741 aactccctag gaaatagaca tttagccagt gttgagaaac ttgcagcatt ggttgaaaat
     1801 gcggaggtga ctagtggatc catgacagtt attcagaagt cgtctcctac agtgaaagga
     1861 gtgaagacaa caacatcaga ttgcaccgaa agacgtcact catcgagttc accttgcaaa
     1921 atgagtcaaa acggaagcag tggatactat gaaggagatg ttttactgga tgatgatgct
     1981 cttgtttcga tgccagaagc aacgctcatt gagtgctttg gtaatctggc tgtcacaaaa
     2041 agacctgcac atctgagacc tccggcagct ttctgtctac cgtttgtatc aatgaacaac
     2101 gatttgacga gcggacatgc cttgaaacat atcacattga catcagtatt cgacgcacta
     2161 catccgtacg ttgccatgcc gtacatcaac agaatggata tatgctctgc aatgaacgta
     2221 tattggatca gaaactgttt ggaagctgaa caatccgatt tatttcatcg attcgctctg
     2281 gaaatgcaag tacatttgag tgcatgtttt ggatgtcgcg ttgtcctgga tatctatgga
     2341 tcaaccagaa acggattcgg tacccgattc tgtgctgttg caatgtcact ttctttttca
     2401 ccgagtccac cttcatgggc aaccaattcg gatcgagtga tgagagctgt tgcgaaggca
     2461 cttgtcgatt ttccgaaagc agttgatgag agatatgtca acgccaaagt tccaattgtc
     2521 agattcagaa gcagtgacat ggatatggaa gcagacatca gttacaagaa tgatttggct
     2581 cttcacaata ctcagctttt gcaacaatac tgcaaatggg atcccgaaag actaccgact
     2641 cttggagttt gggtcaaagc gtgggcgaaa cgaagtggag ttggagatgc atcaaaagga
     2701 tcgctgtctt catatgcttg gattgtgatg cttattcatt atcttcagca agtcgaacca
     2761 attcctgtgt tgccatgtct tcaggaaatg aaccatcaga aaagtgaaaa tgtctacgtc
     2821 caaggttaca acacgtatta ttggaaattc gtagacactg ctcgtacacg acgttgtcgt
     2881 gcttctgtcg ttgatttgtt cgttggattc ttggattact atgccacata ctttgactac
     2941 tccacaaatg ttatccagat ggtgtccaag aaattggaat tcaaaccgga tcgctggtgc
     3001 aagtatccaa tgtgtattgc tgatccgttc gagacggatc ataatttggc acaaggtgtt
     3061 gatatgccaa tgtttgaata cattagatcg tgtatggaac actcgaagaa agtattcacg
     3121 gatcgccgca tgcgctcgga gtttctttcg ggatacgggt tcgacgttga cgaatttgat
     3181 gcaaggcatc gaggcgaaat gaacatggaa atggcgtcac aatttgggga attccttctt
     3241 cacaagtgca taatggtgaa acaagccccg aatcgtcaat tccgtgatcg cagtatgagt
     3301 caatcgacga gcataagcaa cacatcgagt atttcctcgt cgggaggcgg ccgcatcttt
     3361 tacccatacg atgttcctga ctatgcgggc tatccctatg acgtcccgga ctatgcagga
     3421 tcctatccat atgacgttcc agattacgct gctcagtgcg gccgctaacc gcatagggta
     3481 ataactgata taattaaatt gaagctctaa tttgtgagtt tagtatacat gcatttactt
     3541 ataatacagt tttttagttt tgctggccgc atcttctcaa atatgcttcc cagcctgctt
     3601 ttctgtaacg ttcaccctct accttagcat cccttccctt tgcaaatagt cctcttccaa
     3661 caataataat gtcagatcct gtagagacca catcatccac ggttctatac tgttgaccca
     3721 atgcgtctcc cttgtcatct aaacccacac cgggtgtcat aatcaaccaa tcgtaacctt
     3781 catctcttcc acccatgtct ctttgagcaa taaagccgat aacaaaatct ttgtcgctct
     3841 tcgcaatgtc aacagtaccc ttagtatatt ctccagtaga tagggagccc ttgcatgaca
     3901 attctgctaa catcaaaagg cctctaggtt cctttgttac ttcttctgcc gcctgcttca
     3961 aaccgctaac aatacctggg cccaccacac cgtgtgcatt cgtaatgtct gcccattctg
     4021 ctattctgta tacacccgca gagtactgca atttgactgt attaccaatg tcagcaaatt
     4081 ttctgtcttc gaagagtaaa aaattgtact tggcggataa tgcctttagc ggcttaactg
     4141 tgccctccat ggaaaaatca gtcaagatat ccacatgtgt ttttagtaaa caaattttgg
     4201 gacctaatgc ttcaactaac tccagtaatt ccttggtggt acgaacatcc aatgaagcac
     4261 acaagtttgt ttgcttttcg tgcatgatat taaatagctt ggcagcaaca ggactaggat
     4321 gagtagcagc acgttcctta tatgtagctt tcgacatgat ttatcttcgt ttcctgcagg
     4381 tttttgttct gtgcagttgg gttaagaata ctgggcaatt tcatgtttct tcaacactac
     4441 atatgcgtat atataccaat ctaagtctgt gctccttcct tcgttcttcc ttctgttcgg
     4501 agattaccga atcaaaaaaa tttcaaagaa accgctgcag gaattcgaca tctttttcct
     4561 ccatttgtct ttcacttact ctattttatt ttgttttatt ttgatatcta ctcttgtcca
     4621 taacatttat gttcccctat attcattatt tgcttttttc cattacctac tttttgtacc
     4681 tcatctctat ttttctgcaa aaatctttgc gctcttacgc tcttcttatt tctgcaacag
     4741 atagagaatt taacattgtg tatttgaaat catttgactg agaatactgt tgtcatgtct
     4801 ttttacatag aaggaactaa aaaaatttaa aaaaaaaaaa acacaaataa cattatattt
     4861 actgtacata ataaatcaag cctgtttcta accgtttata gaagtagttc ttatatgttc
     4921 atccgattag ggcgtatcca aatgtccatg ccatgacaaa accagttatc tctctaaaaa
     4981 acgattcgaa aaaaatgtca tgaagtgggc tttggtagaa ctttcctggt tgaagttgac
     5041 agatccaaac aatgtaaatg acattaatca agttgtacag aatcaggaac caaataaacc
     5101 catggactga ctcgatttgc aaaaatcctg caacaagccc tagaacaagt gctgagctat
     5161 cttgcacgaa aagtaaacgc ttcttattat caaccacatt cgcagttgcg tttatctgag
     5221 taaatacctc ttcatttgag ctcatttcta tctgtctgcc gctgtctttc tcgaacactg
     5281 cttgagcaat ccaacataag gttaaaatta agttgtattt agttatatct ctactagctt
     5341 catttgtgat tacccgggtt actagaaagc atatagaaag aaaaagaatt ggcgtgaccg
     5401 tacatattgc agcgcattaa atgttctata tcttcatata taataatcaa tgtcattatt
     5461 tcaaataccc acctttctca caaaatcgat accgtcgacc tcgagggggg gcccggtacc
     5521 cagcttttgt tccctttagt gagggttaat tgcgcgcttg gcgtaatcat ggtcatagct
     5581 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccgggagcat
     5641 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     5701 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     5761 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     5821 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     5881 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     5941 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     6001 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     6061 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     6121 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     6181 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     6241 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     6301 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     6361 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     6421 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     6481 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     6541 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     6601 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     6661 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     6721 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     6781 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     6841 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     6901 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     6961 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     7021 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     7081 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     7141 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     7201 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     7261 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     7321 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     7381 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     7441 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     7501 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     7561 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     7621 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     7681 acaaataggg gttccgcgca catttccccg aaaagtgcca c
//
