LOCUS       sequence-467254-        6503 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To display catalytic inactivated rHUH with Y96F mutation on yeast 
            surface.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6503)
  AUTHORS   Huang R, Ting AY
  TITLE     Directed evolution of a sequence-specific covalent protein tag for 
            RNA labeling.
  JOURNAL   Proc Natl Acad Sci U S A. 2025 Mar 4;122(9):e2422085122. doi: 
            10.1073/pnas.2422085122. Epub 2025 Feb 26.
  PUBMED    40009639
REFERENCE   2  (bases 1 to 6503)
  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"; journalName: "Proc Natl 
            Acad Sci U S A."; date: "2025-03-4"; volume: "122(9):e2422085122. 
            doi"; pages: " 10.1073/pnas.2422085122. Epub 2025 Feb 26"
COMMENT     Sequence Label: pCTcon2-rHUH_Y96F
FEATURES             Location/Qualifiers
     source          1..6503
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    202..319
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     614..637
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     CDS             969..980
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             981..1007
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1014..1034
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1449..1478
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(1811..1830)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1812..1830)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1837..1854)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1837..1853)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1846..1868)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      1994..2449
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2081..2100)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2291..2312
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2552..3226)
                     /codon_start=1
                     /gene="S. cerevisiae TRP1"
                     /product="phosphoribosylanthranilate isomerase, required 
                     for tryptophan biosynthesis"
                     /label=TRP1
                     /note="yeast auxotrophic marker"
                     /translation="MSVINFTGSSGPLVKVCGLQSTEAAECALDSDADLLGIICVPNRK
                     RTIDPVIARKISSLVKAYKNSSGTPKYLVGVFRNQPKEDVLALVNDYGIDIVQLHGDES
                     WQEYQEFLGLPVIKRLVFPKDCNILLSAASQKPHSFIPLFDSEAGGTGELLDWNSISDW
                     VGRQESPESLHFMLAGGLTPENVGDALRLNGVIGVDVSGGVETNGVKDSNKIANFVKNA
                     KK"
     promoter        complement(3227..3507)
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     primer_bind     complement(3528..3547)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3647..3669
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3707..3725)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    3766..4269
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        4306..4410
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4411..4479,4480..5271)
                     /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     complement(4629..4648)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5442..6030
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5931..5950
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6184..6201
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6318..6339
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6354..6359,6360..6377,6378..6384)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6392..6408
                     /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)."
     primer_bind     6397..6419
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6416..6432
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6416..6432
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        6453..6471
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        join(6501..6503,1..650)
                     /gene="GAL1 and GAL10 genes of S. cerevisiae"
                     /label=GAL1,10 promoter
                     /note="divergent inducible promoter, regulated by Gal4"
ORIGIN
        1 caaaaattct tacttttttt ttggatggac gcaaagaagt ttaataatca tattacatgg
       61 cattaccacc atatacatat ccatatctaa tcttacttat atgttgtgga aatgtaaaga
      121 gccccattat cttagcctaa aaaaaccttc tctttggaac tttcagtaat acgcttaact
      181 gctcattgct atattgaagt acggattaga agccgccgag cgggtgacag ccctccgaag
      241 gaagactctc ctccgtgcgt cctcgtcttc accggtcgcg ttcctgaaac gcagatgtgc
      301 ctcgcgccgc actgctccga acaataaaga ttctacaata ctagctttta tggttatgaa
      361 gaggaaaaat tggcagtaac ctggccccac aaaccttcaa atgaacgaat caaattaaca
      421 accataggat gataatgcga ttagtttttt agccttattt ctggggtaat taatcagcga
      481 agcgatgatt tttgatctat taacagatat ataaatgcaa aaactgcata accactttaa
      541 ctaatacttt caacattttc ggtttgtatt acttcttatt caaatgtaat aaaagtatca
      601 acaaaaaatt gttaatatac ctctatactt taacgtcaag gagaaaaaac cccggatcga
      661 attccctact tcatacattt tcaattaaga tgcagttact tcgctgtttt tcaatatttt
      721 ctgttattgc ttcagtttta gcacaggaac tgacaactat atgcgagcaa atcccctcac
      781 caactttaga atcgacgccg tactctttgt caacgactac tattttggcc aacgggaagg
      841 caatgcaagg agtttttgaa tattacaaat cagtaacgtt tgtcagtaat tgcggttctc
      901 acccctcaac aactagcaaa ggcagcccca taaacacaca gtatgttttt aaggacaata
      961 gctcgacgat tgaaggtaga tacccatacg acgttccaga ctacgctctg caggagaact
     1021 tgtactttca aggtctgcag gctagtggtg gaggaggctc tggtggaggc ggtagcggag
     1081 gcggagggtc ggctagcccg tcaaagaaaa actggcgcag tgggccgcag ccccacaagc
     1141 gatgggtgtt cacgctgcac catccgtctg aggacgaccg caagaaaatt agggatcttc
     1201 ccatttccct gttcgattac ttcatagttg gcgaagaagg taatgaggag ggacgcacac
     1261 ctcaccttca aggattcgca aacttcgcaa agaaacagac atttcacaag gtgaagtggt
     1321 atttgggtgc ccggtgttgg atagaggagg ccagggggac agaccaacaa aataaagcat
     1381 tttgcagtaa agagggaaac ctcctcaggg agtgcggcgc tccacgatct caaggccggc
     1441 gtggatccga acaaaagctt atttctgaag aggacttgta atagctcgag atctgataac
     1501 aacagtgtag atgtaacaaa atcgactttg ttcccactgt acttttagct cgtacaaaat
     1561 acaatatact tttcatttct ccgtaaacaa catgttttcc catgtaatat ccttttctat
     1621 ttttcgttcc gttaccaact ttacacatac tttatatagc tattcacttc tatacactaa
     1681 aaaactaaga caattttaat tttgctgcct gccatatttc aatttgttat aaattcctat
     1741 aatttatcct attagtagct aaaaaaagat gaatgtgaat cgaatcctaa gagaattgag
     1801 ctccaattcg ccctatagtg agtcgtatta caattcactg gccgtcgttt tacaacgtcg
     1861 tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc
     1921 cagctggcgt aatagcgaag aggcccgcac cgatcgcctt tcccaacagt tgcgcagcct
     1981 gaatggcgaa tggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     2041 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     2101 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     2161 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     2221 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     2281 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     2341 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     2401 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaatttc ctgatgcggt
     2461 attttctcct tacgcatctg tgcggtattt cacaccgcat agatcggcaa gtgcacaaac
     2521 aatacttaaa taaatactac tcagtaataa cctatttctt agcatttttg acgaaatttg
     2581 ctattttgtt agagtctttt acaccatttg tctccacacc tccgcttaca tcaacaccaa
     2641 taacgccatt taatctaagc gcatcaccaa cattttctgg cgtcagtcca ccagctaaca
     2701 taaaatgtaa gctttcgggg ctctcttgcc ttccaaccca gtcagaaatc gagttccaat
     2761 ccaaaagttc acctgtccca cctgcttctg aatcaaacaa gggaataaac gaatgaggtt
     2821 tctgtgaagc tgcactgagt agtatgttgc agtcttttgg aaatacgagt cttttaataa
     2881 ctggcaaacc gaggaactct tggtattctt gccacgactc atctccatgc agttggacga
     2941 tatcaatgcc gtaatcattg accagagcca aaacatcctc cttaggttga ttacgaaaca
     3001 cgccaaccaa gtatttcgga gtgcctgaac tatttttata tgcttttaca agacttgaaa
     3061 ttttccttgc aataaccggg tcaattgttc tctttctatt gggcacacat ataataccca
     3121 gcaagtcagc atcggaatct agagcacatt ctgcggcctc tgtgctctgc aagccgcaaa
     3181 ctttcaccaa tggaccagaa ctacctgtga aattaataac agacatactc caagctgcct
     3241 ttgtgtgctt aatcacgtat actcacgtgc tcaatagtca ccaatgccct ccctcttggc
     3301 cctctccttt tcttttttcg accgaattaa ttcttaatcg gcaaaaaaag aaaagctccg
     3361 gatcaagatt gtacgtaagg tgacaagcta tttttcaata aagaatatct tccactactg
     3421 ccatctggcg tcataactgc aaagtacaca tatattacga tgctgttcta ttaaatgctt
     3481 cctatattat atatatagta atgtcgtgat ctatggtgca ctctcagtac aatctgctct
     3541 gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     3601 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     3661 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc
     3721 ctatttttat aggttaatgt catgataata atggtttctt agacggatcg cttgcctgta
     3781 acttacacgc gcctcgtatc ttttaatgat ggaataattt gggaatttac tctgtgttta
     3841 tttattttta tgttttgtat ttggatttta gaaagtaaat aaagaaggta gaagagttac
     3901 ggaatgaaga aaaaaaaata aacaaaggtt taaaaaattt caacaaaaag cgtactttac
     3961 atatatattt attagacaag aaaagcagat taaatagata tacattcgat taacgataag
     4021 taaaatgtaa aatcacagga ttttcgtgtg tggtcttcta cacagacaag atgaaacaat
     4081 tcggcattaa tacctgagag caggaagagc aagataaaag gtagtatttg ttggcgatcc
     4141 ccctagagtc ttttacatct tcggaaaaca aaaactattt tttctttaat ttcttttttt
     4201 actttctatt tttaatttat atatttatat taaaaaattt aaattataat tatttttata
     4261 gcacgtgatg aaaaggaccc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     4321 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     4381 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     4441 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     4501 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     4561 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     4621 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     4681 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     4741 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     4801 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     4861 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     4921 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
     4981 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     5041 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     5101 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
     5161 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     5221 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     5281 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     5341 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     5401 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     5461 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     5521 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     5581 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     5641 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     5701 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     5761 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     5821 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     5881 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     5941 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     6001 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     6061 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     6121 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
     6181 cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc
     6241 gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc
     6301 agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac
     6361 tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga
     6421 aacagctatg accatgatta cgccaagctc gaaattaacc ctcactaaag ggaacaaaag
     6481 ctggtaccaa ttccttgaat ttt
//
