LOCUS       sequence-466779-        5726 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express catalytic inactivated rHUH-myc protein with Y96F mutation
            in bacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5726)
  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 5726)
  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: pTD68-rHUH_Y96F
FEATURES             Location/Qualifiers
     source          1..5726
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..105
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(106..174,175..966)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(324..343)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1137..1725
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1626..1645
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1879..1896
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    1911..2050
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(1976..1995)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2136..2158
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2152..2343)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3152..4234)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     4196..4215
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4235..4312)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4518..4537
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4621..4640
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4621..4639
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4640..4664
                     /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)."
     RBS             4679..4701
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5062..5091
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(5148..5166)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5162..5209
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      5246..5701
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5333..5352)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5543..5564
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
       61 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
      121 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
      181 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
      241 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
      301 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg
      361 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
      421 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
      481 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
      541 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
      601 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgc agcaatggca
      661 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
      721 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
      781 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca
      841 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
      901 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
      961 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     1021 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     1081 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     1141 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     1201 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     1261 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
     1321 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     1381 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     1441 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     1501 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     1561 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     1621 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     1681 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     1741 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     1801 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     1861 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg
     1921 tattttctcc ttacgcatct gtgcggtatt tcacaccgca atggtgcact ctcagtacaa
     1981 tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt
     2041 catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     2101 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
     2161 ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg
     2221 aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt tctccagaag
     2281 cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt cctgtttggt
     2341 cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac cgatgaaacg
     2401 agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac tggaacgttg
     2461 tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca ctcagggtca
     2521 atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc agcatcctgc
     2581 gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca gactttacga
     2641 aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca
     2701 gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa ggcaaccccg
     2761 ccagcctagc cgggtcctca acgacaggag cacgatcatg cgcacccgtg gggccgccat
     2821 gccggcgata atggcctgct tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc
     2881 ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct
     2941 ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct gccggcacct gtcctacgag
     3001 ttgcatgata aagaagacag tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg
     3061 gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatcccg gtgcctaatg
     3121 agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     3181 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     3241 gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg
     3301 cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat
     3361 cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc
     3421 ccactaccga gatatccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc
     3481 ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca
     3541 tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg
     3601 gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga
     3661 cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct
     3721 ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt
     3781 cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat
     3841 cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt
     3901 gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg
     3961 cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg
     4021 ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca
     4081 cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg
     4141 cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat
     4201 actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt
     4261 ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct
     4321 cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag gttgaggccg
     4381 ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg
     4441 gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga
     4501 gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg
     4561 ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatctcgat cccgcgaaat
     4621 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
     4681 tgtttaactt taagaaggag atataccatg ccgtcaaaga aaaactggcg cagtgggccg
     4741 cagccccaca agcgatgggt gttcacgctg caccatccgt ctgaggacga ccgcaagaaa
     4801 attagggatc ttcccatttc cctgttcgat tacttcatag ttggcgaaga aggtaatgag
     4861 gagggacgca cacctcacct tcaaggattc gcaaacttcg caaagaaaca gacatttcac
     4921 aaggtgaagt ggtatttggg tgcccggtgt tggatagagg aggccagggg gacagaccaa
     4981 caaaataaag cattttgcag taaagaggga aacctcctca gggagtgcgg cgctccacga
     5041 tctcaaggcc ggcgtggatc cgaacaaaag cttatttctg aagaggactt gtaatagctc
     5101 gagggctgct aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata
     5161 actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg
     5221 aactatatcc ggattggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt
     5281 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     5341 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     5401 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     5461 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     5521 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
     5581 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
     5641 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt
     5701 taggtggcac ttttcgggga aatgtg
//
