LOCUS       sequence-436949-        5668 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Base construct used to create PUP2-DADA/3HA fusion proteins in S. 
            cerevisiae. (aka pBSII/PUP2-DADA/3HA/URA3).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5668)
  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 5668)
  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-2
FEATURES             Location/Qualifiers
     source          1..5668
                     /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"
     primer_bind     677..693
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     677..693
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     CDS             2234..2323
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     CDS             complement(2427..3230)
                     /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(3434..3450)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3435..3451)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(3480..3500)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3480..3498)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3519..3535)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3519..3535)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3532..3554)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3543..3559
                     /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(3567..3573,3574..3591,3592..3597))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3612..3633
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3750..3767)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3921..4509)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4001..4020)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4680..5471,5472..5540))
                     /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     5303..5322
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5541..5645)
                     /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 ggccgctcta gaactagtgg atccatgaca gttattcaga agtcgtctcc
      721 tacagtgaaa ggagtgaaga caacaacatc agattgcacc gaaagacgtc actcatcgag
      781 ttcaccttgc aaaatgagtc aaaacggaag cagtggatac tatgaaggag atgttttact
      841 ggatgatgat gctcttgttt cgatgccaga agcaacgctc attgagtgct ttggtaatct
      901 ggctgtcaca aaaagacctg cacatctgag acctccggca gctttctgtc taccgtttgt
      961 atcaatgaac aacgatttga cgagcggaca tgccttgaaa catatcacat tgacatcagt
     1021 attcgacgca ctacatccgt acgttgccat gccgtacatc aacagaatgg atatatgctc
     1081 tgcaatgaac gtatattgga tcagaaactg tttggaagct gaacaatccg atttatttca
     1141 tcgattcgct ctggaaatgc aagtacattt gagtgcatgt tttggatgtc gcgttgtcct
     1201 ggatatctat ggatcaacca gaaacggatt cggtacccga ttctgtgctg ttgcaatgtc
     1261 actttctttt tcaccgagtc caccttcatg ggcaaccaat tcggatcgag tgatgagagc
     1321 tgttgcgaag gcacttgtcg attttccgaa agcagttgat gagagatatg tcaacgccaa
     1381 agttccaatt gtcagattca gaagcagtga catggatatg gaagcagaca tcagttacaa
     1441 gaatgatttg gctcttcaca atactcagct tttgcaacaa tactgcaaat gggatcccga
     1501 aagactaccg actcttggag tttgggtcaa agcgtgggcg aaacgaagtg gagttggaga
     1561 tgcatcaaaa ggatcgctgt cttcatatgc ttggattgtg atgcttattc attatcttca
     1621 gcaagtcgaa ccaattcctg tgttgccatg tcttcaggaa atgaaccatc agaaaagtga
     1681 aaatgtctac gtccaaggtt acaacacgta ttattggaaa ttcgtagaca ctgctcgtac
     1741 acgacgttgt cgtgcttctg tcgttgattt gttcgttgga ttcttggatt actatgccac
     1801 atactttgac tactccacaa atgttatcca gatggtgtcc aagaaattgg aattcaaacc
     1861 ggatcgctgg tgcaagtatc caatgtgtat tgctgatccg ttcgagacgg atcataattt
     1921 ggcacaaggt gttgatatgc caatgtttga atacattaga tcgtgtatgg aacactcgaa
     1981 gaaagtattc acggatcgcc gcatgcgctc ggagtttctt tcgggatacg ggttcgacgt
     2041 tgacgaattt gatgcaaggc atcgaggcga aatgaacatg gaaatggcgt cacaatttgg
     2101 ggaattcctt cttcacaagt gcataatggt gaaacaagcc ccgaatcgtc aattccgtga
     2161 tcgcagtatg agtcaatcga cgagcataag caacacatcg agtatttcct cgtcgggagg
     2221 cggccgcatc ttttacccat acgatgttcc tgactatgcg ggctatccct atgacgtccc
     2281 ggactatgca ggatcctatc catatgacgt tccagattac gctgctcagt gcggccgcta
     2341 accgcatagg gtaataactg atataattaa attgaagctc taatttgtga gtttagtata
     2401 catgcattta cttataatac agttttttag ttttgctggc cgcatcttct caaatatgct
     2461 tcccagcctg cttttctgta acgttcaccc tctaccttag catcccttcc ctttgcaaat
     2521 agtcctcttc caacaataat aatgtcagat cctgtagaga ccacatcatc cacggttcta
     2581 tactgttgac ccaatgcgtc tcccttgtca tctaaaccca caccgggtgt cataatcaac
     2641 caatcgtaac cttcatctct tccacccatg tctctttgag caataaagcc gataacaaaa
     2701 tctttgtcgc tcttcgcaat gtcaacagta cccttagtat attctccagt agatagggag
     2761 cccttgcatg acaattctgc taacatcaaa aggcctctag gttcctttgt tacttcttct
     2821 gccgcctgct tcaaaccgct aacaatacct gggcccacca caccgtgtgc attcgtaatg
     2881 tctgcccatt ctgctattct gtatacaccc gcagagtact gcaatttgac tgtattacca
     2941 atgtcagcaa attttctgtc ttcgaagagt aaaaaattgt acttggcgga taatgccttt
     3001 agcggcttaa ctgtgccctc catggaaaaa tcagtcaaga tatccacatg tgtttttagt
     3061 aaacaaattt tgggacctaa tgcttcaact aactccagta attccttggt ggtacgaaca
     3121 tccaatgaag cacacaagtt tgtttgcttt tcgtgcatga tattaaatag cttggcagca
     3181 acaggactag gatgagtagc agcacgttcc ttatatgtag ctttcgacat gatttatctt
     3241 cgtttcctgc aggtttttgt tctgtgcagt tgggttaaga atactgggca atttcatgtt
     3301 tcttcaacac tacatatgcg tatatatacc aatctaagtc tgtgctcctt ccttcgttct
     3361 tccttctgtt cggagattac cgaatcaaaa aaatttcaaa gaaaccgctg caggaattcg
     3421 atatcaagct tatcgatacc gtcgacctcg agggggggcc cggtacccag cttttgttcc
     3481 ctttagtgag ggttaattgc gcgcttggcg taatcatggt catagctgtt tcctgtgtga
     3541 aattgttatc cgctcacaat tccacacaac atacgagccg ggagcataaa gtgtaaagcc
     3601 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     3661 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     3721 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     3781 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     3841 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     3901 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     3961 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     4021 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     4081 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     4141 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     4201 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     4261 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     4321 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     4381 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     4441 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     4501 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     4561 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     4621 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     4681 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     4741 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     4801 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     4861 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     4921 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     4981 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     5041 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     5101 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     5161 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     5221 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     5281 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     5341 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     5401 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     5461 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     5521 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     5581 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     5641 ccgcgcacat ttccccgaaa agtgccac
//
