LOCUS       sequence-497716-        7889 bp    DNA     circular SYN 16-JAN-2026
DEFINITION  expresses the yeast analog-sensitive Smk1-Q120A and Ssp2 fused to 
            glutathione-S-transferase in bacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7889)
  AUTHORS   Rimal A, Kamdar ZP, Tio CW, Winter E
  TITLE     Isc10, an Inhibitor That Links the Anaphase-Promoting Complex to a 
            Meiosis-Specific Mitogen-Activated Protein Kinase.
  JOURNAL   Mol Cell Biol. 2020 Jul 29;40(16). pii: MCB.00097-20. doi: 
            10.1128/MCB.00097-20. Print 2020 Jul 29.
  PUBMED    32423992
REFERENCE   2  (bases 1 to 7889)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jan 16, 2026 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Mol Cell 
            Biol."; date: "2020-07-29"; volume: "40(16). pii: MCB.00097-20. 
            doi"; pages: " 10.1128/MCB.00097-20. Print 2020 Jul 29"
COMMENT     Sequence Label: pJTB126
FEATURES             Location/Qualifiers
     source          1..7889
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    3..27
                     /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             42..64
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             797..820
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             827..1480
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     1438..1460
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     promoter        1567..1585
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1586..1610
                     /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)."
     CDS             2838..2882
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2920..2938)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2934..2981
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3018..3473
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3105..3124)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3315..3336
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(3588..4379,4380..4448))
                     /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     4211..4230
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4449..4541)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4622..5210
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5111..5130
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5364..5381
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5396..5535
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5461..5480)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5621..5643
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5637..5828)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6400..7482)
                     /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     7444..7463
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7483..7560)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7766..7785
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        join(7873..7889,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag
       61 gagatatacc atgggaaaag tatattcaga agaatcagag gttgttgatg agaaaacggc
      121 aggttctcag aatcccgctc ttctcaaaac aagatcaatt tcattaaacg atataccgcg
      181 cggcacaggc atctctagtg tattgtctca agtacgcgga ggttccttag aaagaattat
      241 tgtctatagg tacgatactc cggaacgttc tttgcataaa gttgatcttt tcttcttaaa
      301 ttacgaggga gctcaatctt ttatgagata cgcaaaaaca aatattttta aagttaatgg
      361 agttcaatta aaaccagaat ggatattcct cgagagcaca tacgaaaaca taatgaaaga
      421 gcaatctgtc aacagaataa tagaagagga aaagttcata tcaagatgtt tgatagtgaa
      481 aaaaagctcg acaacagcaa tgcctaacaa atcaaatctg aataaagggc aaactttaga
      541 aaacattgat attcaagaat tagaaaaaga ttttcagaat tttggagagg ttctagaaat
      601 tacgcctatt gtatcaagaa aattatgcgt atctatattc ttttacgata tttccagtgc
      661 tatgagagct atggaagaat acgaacaaaa gggctcatat ctatataata aatattttaa
      721 aacatggaca atatggtacg ggaaagatat tacagaccag ccttgcattg acttgcggat
      781 ccccgggtta attaatctgg aagttctgtt ccaggggccc ggcggcatgt cccctatact
      841 aggttattgg aaaattaagg gccttgtgca acccactcga cttcttttgg aatatcttga
      901 agaaaaatat gaagagcatt tgtatgagcg cgatgaaggt gataaatggc gaaacaaaaa
      961 gtttgaattg ggtttggagt ttcccaatct tccttattat attgatggtg atgttaaatt
     1021 aacacagtct atggccatca tacgttatat agctgacaag cacaacatgt tgggtggttg
     1081 tccaaaagag cgtgcagaga tttcaatgct tgaaggagcg gttttggata ttagatacgg
     1141 tgtttcgaga attgcatata gtaaagactt tgaaactctc aaagttgatt ttcttagcaa
     1201 gctacctgaa atgctgaaaa tgttcgaaga tcgtttatgt cataaaacat atttaaatgg
     1261 tgatcatgta acccatcctg acttcatgtt gtatgacgct cttgatgttg ttttatacat
     1321 ggacccaatg tgcctggatg cgttcccaaa attagtttgt tttaaaaaac gtattgaagc
     1381 tatcccacaa attgataagt acttgaaatc cagcaagtat atagcatggc ctttgcaggg
     1441 ctggcaagcc acgtttggtg gtggcgacca tcctccaaaa tcggatctgg ttccgcgttg
     1501 agcggccgca taatgcttaa gtcgaacaga aagtaatcgt attgtacacg gccgcataat
     1561 cgaaattaat acgactcact ataggggaat tgtgagcgga taacaattcc ccatcttagt
     1621 atattagtta agtataagaa ggagatatac atatgaatcg cacacttaca gataatacca
     1681 gagccataaa tgtggcctcc aacctcggtg ctcctcaaca gaggacgatt tttgccaagg
     1741 agagaatatc catcccagga tactacgaga taatccaatt cctgggcaag ggagcctatg
     1801 ggacagtgtg ctcagtgaag tttaagggtc gaagcccagc tgctaggata gcggtaaaga
     1861 agatcagcaa catctttaat aaggagatcc ttttgaaaag ggccatccgt gagctaaaat
     1921 tcatgaactt cttcaagggc cataaaaata ttgtaaatct aatcgatcta gagatagtga
     1981 ccagctcgcc ctatgacgga ctgtattgct acgctgaact gattgattat gatttggcaa
     2041 aagtcataca ttcgtccgtg cagctctcgg agtttcatat caaatacttt ctttaccaaa
     2101 tcctgtgtgg gctaaagtac atacacagtg cagatgtcat ccaccgagat ttgaagcctg
     2161 ggaacatatt gtgcacattg aatggctgtt tgaagatctg cgattttggt ctcgctaggg
     2221 gtattcatgc cggatttttc aagtgtcatt ccactgtgca accccatata accaattacg
     2281 ttgctacaag atggtacagg gcgcctgagc tgcttctttc caaccaaccg tatagtaagt
     2341 ccgtagacat ctgggctgta gggtgcattc ttgcagaatt ctatgctcga aagcctgttt
     2401 tcatggggcg tgactccatg caccagatat ttgaaatcat caaagtcttg ggtactcctg
     2461 ataaagatat tctgatcaag tttggcacca ttaaagcttg gaatctaggc aaaaacagta
     2521 ataaccctgt atataaaaaa atcccatggt ccaacatttt cccctttgcc tcgcatgaag
     2581 caatcaatct catagagtca ttacttcatt gggactcaac acataggtta aacgtggaac
     2641 aagctatatc acatccgttc ctaaacgagg tgcggaagcc agacgacgag cctgtttgcc
     2701 ttcagggtcc tttcgacttc acctatgaat ccgagttgaa ttcaatgtct aaattaagag
     2761 actacttggt tgaagaagtg aaaaatttca aaaccgattt gtcctcctcg tctttatgag
     2821 gtaccctcga gtctggtaaa gaaaccgctg ctgcgaaatt tgaacgccag cacatggact
     2881 cgtctactag cgcagcttaa ttaacctagg ctgctgccac cgctgagcaa taactagcat
     2941 aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga ggaactatat
     3001 ccggattggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     3061 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     3121 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     3181 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     3241 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     3301 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     3361 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     3421 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa tttctggcgg
     3481 cacgatggca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     3541 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     3601 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     3661 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     3721 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     3781 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     3841 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     3901 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     3961 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     4021 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     4081 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     4141 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     4201 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     4261 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     4321 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     4381 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     4441 atactcatac tcttcctttt tcaatcatga ttgaagcatt tatcagggtt attgtctcat
     4501 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggtcatg accaaaatcc
     4561 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     4621 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     4681 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     4741 tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact
     4801 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     4861 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     4921 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     4981 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     5041 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     5101 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     5161 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     5221 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     5281 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     5341 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga
     5401 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcaatggt gcactctcag
     5461 tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac
     5521 tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     5581 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     5641 aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg
     5701 tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc
     5761 agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt
     5821 ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg
     5881 aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa
     5941 cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag
     6001 ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat
     6061 cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt
     6121 tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga cgttttgcag
     6181 cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa
     6241 ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgctagt catgccccgc
     6301 gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg agatcccggt
     6361 gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg
     6421 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     6481 cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc
     6541 cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag
     6601 gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc
     6661 gtcgtatccc actaccgaga tgtccgcacc aacgcgcagc ccggactcgg taatggcgcg
     6721 cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc
     6781 attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc
     6841 cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg
     6901 cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac
     6961 cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg
     7021 tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc
     7081 aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag
     7141 aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac
     7201 cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc
     7261 gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg
     7321 ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg
     7381 cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac
     7441 accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg
     7501 actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc
     7561 cgggatctcg acgctctccc ttatgcgact cctgcattag gaagcagccc agtagtaggt
     7621 tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg caaggagatg gcgcccaaca
     7681 gtcccccggc cacggggcct gccaccatac ccacgccgaa acaagcgctc atgagcccga
     7741 agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac
     7801 ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggatcgag atcgatctcg
     7861 atcccgcgaa attaatacga ctcactata
//
