LOCUS       sequence-392969-        6312 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression plasmid for gamma toxin tRNA endonuclease.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6312)
  AUTHORS   Lentini JM, Ramos J, Fu D
  TITLE     Monitoring the 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) 
            modification in eukaryotic tRNAs via the gamma-toxin endonuclease.
  JOURNAL   RNA. 2018 May;24(5):749-758. doi: 10.1261/rna.065581.118. Epub 2018 
            Feb 13.
  PUBMED    29440318
REFERENCE   2  (bases 1 to 6312)
  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: 
            "10.1261/rna.065581.118"; journalName: "RNA"; date: "2018-05"; 
            volume: "24"; issue: "5"; pages: "749-758"
COMMENT     Sequence Label: pET28-His10Smt3-gamma-toxin
FEATURES             Location/Qualifiers
     source          1..6312
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     113..130
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    145..284
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(210..229)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     370..392
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(386..577)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1386..2468)
                     /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     2430..2449
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2469..2546)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2752..2771
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2855..2874
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2855..2873
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2874..2898
                     /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             2913..2935
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2948..2977
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             2990..3001
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             3014..3307
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             4031..4048
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4101..4119)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4115..4162
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4199..4654
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4286..4305)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4496..4517
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4746..5561)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(4746..5561)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     5469..5488
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5553..5572
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5683..6271
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6172..6191
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
       61 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
      121 agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg
      181 tatttcacac cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
      241 cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa
      301 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
      361 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
      421 ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt
      481 catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc
      541 gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat
      601 ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta
      661 ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga
      721 tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg
      781 taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc
      841 agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg
      901 ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg
      961 gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca
     1021 ggagcacgat catgcgcacc cgtggggccg ccatgccggc gataatggcc tgcttctcgc
     1081 cgaaacgttt ggtggcggga ccagtgacga aggcttgagc gagggcgtgc aagattccga
     1141 ataccgcaag cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa
     1201 tgacccagag cgctgccggc acctgtccta cgagttgcat gataaagaag acagtcataa
     1261 gtgcggcgac gatagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc
     1321 tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca ttaattgcgt
     1381 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     1441 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc
     1501 agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag ttgcagcaag
     1561 cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt taacggcggg
     1621 atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg
     1681 cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc
     1741 agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg aaaaccggac
     1801 atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg agtgagatat
     1861 ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc cgctaacagc
     1921 gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca
     1981 tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa taacgccgga
     2041 acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg atagttaatg
     2101 atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg
     2161 ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc gcgagattta
     2221 atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc aacgccaatc
     2281 agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta attcagctcc
     2341 gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc
     2401 acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta taacgttact
     2461 ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc cataccgcga
     2521 aaggttttgc gccattcgat ggtgtccggg atctcgacgc tctcccttat gcgactcctg
     2581 cattaggaag cagcccagta gtaggttgag gccgttgagc accgccgccg caaggaatgg
     2641 tgcatgcaag gagatggcgc ccaacagtcc cccggccacg gggcctgcca ccatacccac
     2701 gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga tcttccccat cggtgatgtc
     2761 ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc
     2821 ggcgtagagg atcgagatct cgatcccgcg aaattaatac gactcactat aggggaattg
     2881 tgagcggata acaattcccc tctagaaata attttgttta actttaagaa ggagatatac
     2941 catgggccat catcatcatc atcatcatca tcatcacagc agcggccata tcgaaggtcg
     3001 tcatatggct agcatgtcgg actcagaagt caatcaagaa gctaagccag aggtcaagcc
     3061 agaagtcaag cctgagactc acatcaattt aaaggtgtcc gatggatctt cagagatctt
     3121 cttcaagatc aaaaagacca ctcctttaag aaggctgatg gaagcgttcg ctaaaagaca
     3181 gggtaaggaa atggactcct taagattctt gtacgacggt attagaattc aagctgatca
     3241 gacccctgaa gatttggaca tggaggataa cgatattatt gaggctcaca gagaacagat
     3301 tggtggatcc atggctgcaa ccactgcccg tgaagaattt ttcctgtgtt acgatttgat
     3361 cagatacttg aagcaatacg aaaagactgg tgaatctaaa ctggttgaac agacgttttt
     3421 caactccatc aagaacttgg atatcaattc tcgcgaatat atggaactgg tgtacaacaa
     3481 aattgctggt atcagtaacg aacgtaacaa gttcgaaaac atctataaag atggcgattc
     3541 tatctcacaa gttgtggaac gtgcagtgtc cgaaaagaaa ctgaccttcg gtttaaacgg
     3601 taaaggctta tatgtcccag aaaatggtga accgagattg aaaggctacg catcaattat
     3661 cgaacgcatt actctggatt taatggaaat ttacagcatc aaaggcctga acgatattcc
     3721 tagagatatc aagttcaaca tggaaaagat tcgccaagaa cgttataacc agatgaaaga
     3781 agccctgaat tctgtcgaag gttacaaagg caaaatcgtt gcgagcgata gtgattggtg
     3841 ctttaaagat ccacaaggta accgcatcac agatttcgat tcaatcaaca aggaattggg
     3901 tctgggccgt agagatgtca aactggataa aggtcatgat gatttgatca aattgtgtac
     3961 tgaaaagatc gattctatga acaacttgca gaacggcaag tgcgtgtaaa agcttgcggc
     4021 cgcactcgag caccaccacc accaccactg agatccggct gctaacaaag cccgaaagga
     4081 agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa
     4141 acgggtcttg aggggttttt tgctgaaagg aggaactata tccggattgg cgaatgggac
     4201 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     4261 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     4321 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     4381 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca
     4441 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     4501 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
     4561 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     4621 gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc
     4681 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgaatt
     4741 aattcttaga aaaactcatc gagcatcaaa tgaaactgca atttattcat atcaggatta
     4801 tcaataccat atttttgaaa aagccgtttc tgtaatgaag gagaaaactc accgaggcag
     4861 ttccatagga tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc aacatcaata
     4921 caacctatta atttcccctc gtcaaaaata aggttatcaa gtgagaaatc accatgagtg
     4981 acgactgaat ccggtgagaa tggcaaaagt ttatgcattt ctttccagac ttgttcaaca
     5041 ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt
     5101 gattgcgcct gagcgagacg aaatacgcga tcgctgttaa aaggacaatt acaaacagga
     5161 atcgaatgca accggcgcag gaacactgcc agcgcatcaa caatattttc acctgaatca
     5221 ggatattctt ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat
     5281 gcatcatcag gagtacggat aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc
     5341 cagtttagtc tgaccatctc atctgtaaca tcattggcaa cgctaccttt gccatgtttc
     5401 agaaacaact ctggcgcatc gggcttccca tacaatcgat agattgtcgc acctgattgc
     5461 ccgacattat cgcgagccca tttataccca tataaatcag catccatgtt ggaatttaat
     5521 cgcggcctag agcaagacgt ttcccgttga atatggctca taacacccct tgtattactg
     5581 tttatgtaag cagacagttt tattgttcat gaccaaaatc ccttaacgtg agttttcgtt
     5641 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     5701 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     5761 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     5821 aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     5881 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     5941 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     6001 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     6061 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     6121 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     6181 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     6241 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     6301 cctggccttt tg
//
