LOCUS       sequence-436941-        7721 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Construct used to generate the PUF3/PUP2/3HA tagging strains. 
            Surrounding the PUP2/3HA/URA3 are large regions of homology to 
            integrate at the 3' end of the PUF3 gene..
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-4
FEATURES             Location/Qualifiers
     source          1..7721
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(659..675)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(660..676)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(705..725)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(705..723)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(744..760)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(744..760)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(757..779)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    768..784
                     /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(792..798,799..816,817..822))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    837..858
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(975..992)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1146..1734)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1226..1245)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1905..2696,2697..2765))
                     /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     2528..2547
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2766..2870)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2896..3351)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3033..3054)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3245..3264
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3478..3500
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3492..3509
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3493..3509
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3519..3538
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3519..3537
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             6254..6343
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     CDS             complement(6447..7250)
                     /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"
ORIGIN
        1 aaaaaaaaaa aaacacaaat aacattatat ttactgtaca taataaatca agcctgtttc
       61 taaccgttta tagaagtagt tcttatatgt tcatccgatt agggcgtatc caaatgtcca
      121 tgccatgaca aaaccagtta tctctctaaa aaacgattcg aaaaaaatgt catgaagtgg
      181 gctttggtag aactttcctg gttgaagttg acagatccaa acaatgtaaa tgacattaat
      241 caagttgtac agaatcagga accaaataaa cccatggact gactcgattt gcaaaaatcc
      301 tgcaacaagc cctagaacaa gtgctgagct atcttgcacg aaaagtaaac gcttcttatt
      361 atcaaccaca ttcgcagttg cgtttatctg agtaaatacc tcttcatttg agctcatttc
      421 tatctgtctg ccgctgtctt tctcgaacac tgcttgagca atccaacata aggttaaaat
      481 taagttgtat ttagttatat ctctactagc ttcatttgtg attacccggg ttactagaaa
      541 gcatatagaa agaaaaagaa ttggcgtgac cgtacatatt gcagcgcatt aaatgttcta
      601 tatcttcata tataataatc aatgtcatta tttcaaatac ccacctttct cacaaaatcg
      661 ataccgtcga cctcgagggg gggcccggta cccagctttt gttcccttta gtgagggtta
      721 attgcgcgct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
      781 acaattccac acaacatacg agccgggagc ataaagtgta aagcctgggg tgcctaatga
      841 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
      901 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
      961 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     1021 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     1081 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     1141 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     1201 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     1261 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     1321 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     1381 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     1441 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     1501 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     1561 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     1621 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     1681 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     1741 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     1801 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     1861 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     1921 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     1981 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     2041 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     2101 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     2161 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     2221 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     2281 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     2341 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     2401 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     2461 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     2521 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     2581 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     2641 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     2701 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     2761 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     2821 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     2881 cgaaaagtgc cacctaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt
     2941 gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa
     3001 aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa
     3061 agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac
     3121 gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga
     3181 accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa
     3241 aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc
     3301 tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg tcccattcgc
     3361 cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc
     3421 agctggcgaa agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc
     3481 agtcacgacg ttgtaaaacg acggccagtg agcgcgcgta atacgactca ctatagggcg
     3541 aattggagct ccaccgcggt ggcggccgcc tcaaagtcta cagcaacagc agcaaacgta
     3601 tcatcgatcg ccgttgttag agcaactaag aaacagtagt tctgacaaaa attcaaactc
     3661 gaacatgtca ctaaaagaca tctttggcca ttccttagag ttttgcaaag atcaacatgg
     3721 ttcacggttc attcagcgcg agttggccac ctcaccagcc tcagaaaagg aggtcatatt
     3781 caatgaaatt cgcgatgacg ccattgagct ttcgaacgat gtatttggta attacgtcat
     3841 tcagaagttt tttgaatttg gtagcaaaat tcaaaaaaat actttggttg atcaatttaa
     3901 aggtaacatg aaacagttat ctctacaaat gtacgcatgt cgtgtaattc aaaaggcgtt
     3961 agaatatatt gattccaatc aaagaattga gttagtcctg gaattatctg actccgtttt
     4021 gcaaatgatc aaagatcaga acggtaatca cgtaattcaa aaggccatcg aaacaattcc
     4081 tatcgaaaaa ttacctttta tcttaagttc tttaactggc cacatttacc atctatctac
     4141 ccactcctac ggctgtaggg tcattcaaag attgttagaa tttggttcga gcgaagatca
     4201 ggaaagcatt ttaaacgaat tgaaagattt cataccatat ctaattcaag accaatatgg
     4261 taactatgtc attcaatatg ttttgcaaca agatcagttc accaataagg aaatggtcga
     4321 tatcaaacag gaaatcatag aaactgtggc caataatgtt gtcgaatact ctaaacataa
     4381 atttgcctcc aacgtggtcg aaaaatctat tctttatggc tctaaaaatc agaaggattt
     4441 aataatttct aagattttac caagagacaa aaatcatgct ttgaatctgg aagacgattc
     4501 tccaatgatt ttaatgatca aggatcaatt tgccaactat gtgattcaaa aattagttaa
     4561 tgtctctgag ggtgagggta agaaattgat cgtcattgcc attagagcat atttggataa
     4621 actaaacaaa tctaactccc taggaaatag acatttagcc agtgttgaga aacttgcagc
     4681 attggttgaa aatgcggagg tgactagtgg atccatgaca gttattcaga agtcgtctcc
     4741 tacagtgaaa ggagtgaaga caacaacatc agattgcacc gaaagacgtc actcatcgag
     4801 ttcaccttgc aaaatgagtc aaaacggaag cagtggatac tatgaaggag atgttttact
     4861 ggatgatgat gctcttgttt cgatgccaga agcaacgctc attgagtgct ttggtaatct
     4921 ggctgtcaca aaaagacctg cacatctgag acctccggca gctttctgtc taccgtttgt
     4981 atcaatgaac aacgatttga cgagcggaca tgccttgaaa catatcacat tgacatcagt
     5041 attcgacgca ctacatccgt acgttgccat gccgtacatc aacagaatgg atatatgctc
     5101 tgcaatgaac gtatattgga tcagaaactg tttggaagct gaacaatccg atttatttca
     5161 tcgattcgct ctggaaatgc aagtacattt gagtgcatgt tttggatgtc gcgttgtcct
     5221 ggatatctat ggatcaacca gaaacggatt cggtacccga ttctgtgatg ttgatatgtc
     5281 actttctttt tcaccgagtc caccttcatg ggcaaccaat tcggatcgag tgatgagagc
     5341 tgttgcgaag gcacttgtcg attttccgaa agcagttgat gagagatatg tcaacgccaa
     5401 agttccaatt gtcagattca gaagcagtga catggatatg gaagcagaca tcagttacaa
     5461 gaatgatttg gctcttcaca atactcagct tttgcaacaa tactgcaaat gggatcccga
     5521 aagactaccg actcttggag tttgggtcaa agcgtgggcg aaacgaagtg gagttggaga
     5581 tgcatcaaaa ggatcgctgt cttcatatgc ttggattgtg atgcttattc attatcttca
     5641 gcaagtcgaa ccaattcctg tgttgccatg tcttcaggaa atgaaccatc agaaaagtga
     5701 aaatgtctac gtccaaggtt acaacacgta ttattggaaa ttcgtagaca ctgctcgtac
     5761 acgacgttgt cgtgcttctg tcgttgattt gttcgttgga ttcttggatt actatgccac
     5821 atactttgac tactccacaa atgttatcca gatggtgtcc aagaaattgg aattcaaacc
     5881 ggatcgctgg tgcaagtatc caatgtgtat tgctgatccg ttcgagacgg atcataattt
     5941 ggcacaaggt gttgatatgc caatgtttga atacattaga tcgtgtatgg aacactcgaa
     6001 gaaagtattc acggatcgcc gcatgcgctc ggagtttctt tcgggatacg ggttcgacgt
     6061 tgacgaattt gatgcaaggc atcgaggcga aatgaacatg gaaatggcgt cacaatttgg
     6121 ggaattcctt cttcacaagt gcataatggt gaaacaagcc ccgaatcgtc aattccgtga
     6181 tcgcagtatg agtcaatcga cgagcataag caacacatcg agtatttcct cgtcgggagg
     6241 cggccgcatc ttttacccat acgatgttcc tgactatgcg ggctatccct atgacgtccc
     6301 ggactatgca ggatcctatc catatgacgt tccagattac gctgctcagt gcggccgcta
     6361 accgcatagg gtaataactg atataattaa attgaagctc taatttgtga gtttagtata
     6421 catgcattta cttataatac agttttttag ttttgctggc cgcatcttct caaatatgct
     6481 tcccagcctg cttttctgta acgttcaccc tctaccttag catcccttcc ctttgcaaat
     6541 agtcctcttc caacaataat aatgtcagat cctgtagaga ccacatcatc cacggttcta
     6601 tactgttgac ccaatgcgtc tcccttgtca tctaaaccca caccgggtgt cataatcaac
     6661 caatcgtaac cttcatctct tccacccatg tctctttgag caataaagcc gataacaaaa
     6721 tctttgtcgc tcttcgcaat gtcaacagta cccttagtat attctccagt agatagggag
     6781 cccttgcatg acaattctgc taacatcaaa aggcctctag gttcctttgt tacttcttct
     6841 gccgcctgct tcaaaccgct aacaatacct gggcccacca caccgtgtgc attcgtaatg
     6901 tctgcccatt ctgctattct gtatacaccc gcagagtact gcaatttgac tgtattacca
     6961 atgtcagcaa attttctgtc ttcgaagagt aaaaaattgt acttggcgga taatgccttt
     7021 agcggcttaa ctgtgccctc catggaaaaa tcagtcaaga tatccacatg tgtttttagt
     7081 aaacaaattt tgggacctaa tgcttcaact aactccagta attccttggt ggtacgaaca
     7141 tccaatgaag cacacaagtt tgtttgcttt tcgtgcatga tattaaatag cttggcagca
     7201 acaggactag gatgagtagc agcacgttcc ttatatgtag ctttcgacat gatttatctt
     7261 cgtttcctgc aggtttttgt tctgtgcagt tgggttaaga atactgggca atttcatgtt
     7321 tcttcaacac tacatatgcg tatatatacc aatctaagtc tgtgctcctt ccttcgttct
     7381 tccttctgtt cggagattac cgaatcaaaa aaatttcaaa gaaaccgctg caggaattcg
     7441 acatcttttt cctccatttg tctttcactt actctatttt attttgtttt attttgatat
     7501 ctactcttgt ccataacatt tatgttcccc tatattcatt atttgctttt ttccattacc
     7561 tactttttgt acctcatctc tatttttctg caaaaatctt tgcgctctta cgctcttctt
     7621 atttctgcaa cagatagaga atttaacatt gtgtatttga aatcatttga ctgagaatac
     7681 tgttgtcatg tctttttaca tagaaggaac taaaaaaatt t
//
