LOCUS       sequence-476823-        5840 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the tRNAIle intron bearing the 5' GGGAGA initially 
            transcribed sequence (ITS); Plasmid #2 of the tet-on overexpression 
            system.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5840)
  AUTHORS   Nostramo RT, Sinopoli PL, Bao A, Metcalf S, Peltier LM, Hopper AK
  TITLE     Free introns of tRNAs as complementarity-dependent regulators of 
            gene expression.
  JOURNAL   Mol Cell. 2025 Feb 20;85(4):726-741.e6. doi: 
            10.1016/j.molcel.2025.01.019. Epub 2025 Feb 11.
  PUBMED    39938518
REFERENCE   2  (bases 1 to 5840)
  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: 
            "10.1016/j.molcel.2025.01.019"; journalName: "Mol Cell"; date: 
            "2025-02-20- 20"; volume: "85"; issue: "4"; pages: "726-741.e6"
COMMENT     Sequence Label: T7pro-tRNA-ITSIle intron-T7tVar1-pRS426
FEATURES             Location/Qualifiers
     source          1..5840
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        194..409
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             410..1213
                     /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"
     rep_origin      complement(1344..1799)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1481..1502)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1693..1712
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1925..1947
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1939..1956
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1940..1956
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        1966..1984
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        2042..2060
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2185..2201)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2185..2201)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(2238..2258)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2238..2256)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2277..2293)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2277..2293)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2290..2312)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2301..2317
                     /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(2325..2331,2332..2349,2350..2355))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2370..2391
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2508..2525)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2679..3267)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2759..2778)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3438..4229,4230..4298))
                     /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     4061..4080
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4299..4403)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4430..5772
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(5393..5440)
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     primer_bind     5813..5831
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accataccac cttttcaatt catcattttt tttttattct tttttttgat ttcggtttcc
      241 ttgaaatttt tttgattcgg taatctccga acagaaggaa gaacgaagga aggagcacag
      301 acttagattg gtatatatac gcatatgtag tgttgaagaa acatgaaatt gcccagtatt
      361 cttaacccaa ctgcacagaa caaaaacctg caggaaacga agataaatca tgtcgaaagc
      421 tacatataag gaacgtgctg ctactcatcc tagtcctgtt gctgccaagc tatttaatat
      481 catgcacgaa aagcaaacaa acttgtgtgc ttcattggat gttcgtacca ccaaggaatt
      541 actggagtta gttgaagcat taggtcccaa aatttgttta ctaaaaacac atgtggatat
      601 cttgactgat ttttccatgg agggcacagt taagccgcta aaggcattat ccgccaagta
      661 caatttttta ctcttcgaag acagaaaatt tgctgacatt ggtaatacag tcaaattgca
      721 gtactctgcg ggtgtataca gaatagcaga atgggcagac attacgaatg cacacggtgt
      781 ggtgggccca ggtattgtta gcggtttgaa gcaggcggca gaagaagtaa caaaggaacc
      841 tagaggcctt ttgatgttag cagaattgtc atgcaagggc tccctatcta ctggagaata
      901 tactaagggt actgttgaca ttgcgaagag cgacaaagat tttgttatcg gctttattgc
      961 tcaaagagac atgggtggaa gagatgaagg ttacgattgg ttgattatga cacccggtgt
     1021 gggtttagat gacaagggag acgcattggg tcaacagtat agaaccgtgg atgatgtggt
     1081 ctctacagga tctgacatta ttattgttgg aagaggacta tttgcaaagg gaagggatgc
     1141 taaggtagag ggtgaacgtt acagaaaagc aggctgggaa gcatatttga gaagatgcgg
     1201 ccagcaaaac taaaaaactg tattataagt aaatgcatgt atactaaact cacaaattag
     1261 agcttcaatt taattatatc agttattacc ctatgcggtg tgaaataccg cacagatgcg
     1321 taaggagaaa ataccgcatc aggaaattgt aagcgttaat attttgttaa aattcgcgtt
     1381 aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta
     1441 taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc
     1501 actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg
     1561 cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact
     1621 aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt
     1681 ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc
     1741 ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc
     1801 cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta
     1861 ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg
     1921 ttttcccagt cacgacgttg taaaacgacg gccagtgagc gcgcgtaata cgactcacta
     1981 tagggcgaat tgggtaccgg gccccccctc gaggtcgacc tcgagggtac cgccgggaat
     2041 ttaatacgac tcactatagg gagagcaaca ttcggtttcc gaagtttctg tgccaaagac
     2101 ctttcaaaca ggcctttaaa agcaatctgt tttttttggt cgacggtatc gataagcttg
     2161 atatcgaatt cctgcagccc gggggatcca ctagttctag agcggccgcc accgcggtgg
     2221 agctccagct tttgttccct ttagtgaggg ttaattgcgc gcttggcgta atcatggtca
     2281 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     2341 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     2401 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     2461 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     2521 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     2581 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     2641 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     2701 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     2761 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     2821 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     2881 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     2941 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     3001 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     3061 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     3121 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     3181 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     3241 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     3301 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     3361 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     3421 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     3481 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     3541 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     3601 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     3661 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     3721 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     3781 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     3841 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     3901 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     3961 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     4021 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     4081 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     4141 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     4201 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     4261 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     4321 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     4381 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga acgaagcatc
     4441 tgtgcttcat tttgtagaac aaaaatgcaa cgcgagagcg ctaatttttc aaacaaagaa
     4501 tctgagctgc atttttacag aacagaaatg caacgcgaaa gcgctatttt accaacgaag
     4561 aatctgtgct tcatttttgt aaaacaaaaa tgcaacgcga gagcgctaat ttttcaaaca
     4621 aagaatctga gctgcatttt tacagaacag aaatgcaacg cgagagcgct attttaccaa
     4681 caaagaatct atacttcttt tttgttctac aaaaatgcat cccgagagcg ctatttttct
     4741 aacaaagcat cttagattac tttttttctc ctttgtgcgc tctataatgc agtctcttga
     4801 taactttttg cactgtaggt ccgttaaggt tagaagaagg ctactttggt gtctattttc
     4861 tcttccataa aaaaagcctg actccacttc ccgcgtttac tgattactag cgaagctgcg
     4921 ggtgcatttt ttcaagataa aggcatcccc gattatattc tataccgatg tggattgcgc
     4981 atactttgtg aacagaaagt gatagcgttg atgattcttc attggtcaga aaattatgaa
     5041 cggtttcttc tattttgtct ctatatacta cgtataggaa atgtttacat tttcgtattg
     5101 ttttcgattc actctatgaa tagttcttac tacaattttt ttgtctaaag agtaatacta
     5161 gagataaaca taaaaaatgt agaggtcgag tttagatgca agttcaagga gcgaaaggtg
     5221 gatgggtagg ttatataggg atatagcaca gagatatata gcaaagagat acttttgagc
     5281 aatgtttgtg gaagcggtat tcgcaatatt ttagtagctc gttacagtcc ggtgcgtttt
     5341 tggttttttg aaagtgcgtc ttcagagcgc ttttggtttt caaaagcgct ctgaagttcc
     5401 tatactttct agagaatagg aacttcggaa taggaacttc aaagcgtttc cgaaaacgag
     5461 cgcttccgaa aatgcaacgc gagctgcgca catacagctc actgttcacg tcgcacctat
     5521 atctgcgtgt tgcctgtata tatatataca tgagaagaac ggcatagtgc gtgtttatgc
     5581 ttaaatgcgt acttatatgc gtctatttat gtaggatgaa aggtagtcta gtacctcctg
     5641 tgatattatc ccattccatg cggggtatcg tatgcttcct tcagcactac cctttagctg
     5701 ttctatatgc tgccactcct caattggatt agtctcatcc ttcaatgcta tcatttcctt
     5761 tgatattgga tcatactaag aaaccattat tatcatgaca ttaacctata aaaataggcg
     5821 tatcacgagg ccctttcgtc
//
