LOCUS       sequence-497707-        6422 bp    DNA     circular SYN 16-JAN-2026
DEFINITION  Expresses yeast Smk1in bacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6422)
  AUTHORS   Tio CW, Omerza G, Phillips T, Lou HJ, Turk BE, Winter E
  TITLE     Ssp2 Binding Activates the Smk1 Mitogen-Activated Protein Kinase.
  JOURNAL   Mol Cell Biol. 2017 May 2;37(10):e00607-16. doi: 
            10.1128/MCB.00607-16. Print 2017 May 15.
  PUBMED    28223369
REFERENCE   2  (bases 1 to 6422)
  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"; doi: 
            "10.1128/MCB.00607-16"; journalName: "Mol Cell Biol"; date: 
            "2017-05-2- 2"; volume: "37"; issue: "10"; pages: "e00607-16"
COMMENT     Sequence Label: pJTB72
FEATURES             Location/Qualifiers
     source          1..6422
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     69..87
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1359..1381
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1396..1420
                     /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     complement(1420..1439)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1421..1439)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1527..1546)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1752..1829
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1830..2912
                     /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     complement(1849..1868)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3721..3912
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3906..3928)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4014..4153
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4069..4088
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4168..4185)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4339..4927)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4419..4438)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5038..5057)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5049..5864
                     /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             5049..5864
                     /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     complement(5122..5141)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5956..6411)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6093..6114)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6305..6324
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcaagcttgt
      181 cgactcataa agacgaggag gacaaatcgg ttttgaaatt tttcacttct tcaaccaagt
      241 agtctcttaa tttagacatt gaattcaact cggattcata ggtgaagtcg aaaggaccct
      301 gaaggcaaac aggctcgtcg tctggcttcc gcacctcgtt taggaacgga tgtgatatag
      361 cttgttccac gtttaaccta tgtgttgagt cccaatgaag taatgactct atgagattga
      421 ttgcttcatg cgaggcaaag gggaaaatgt tggaccatgg gattttttta tatacagggt
      481 tattactgtt tttgcctaga ttccaagctt taatggtgcc aaacttgatc agaatatctt
      541 tatcaggagt acccaagact ttgatgattt caaatatctg gtgcatggag tcacgcccca
      601 tgaaaacagg ctttcgagca tagaattctg caagaatgca ccctacagcc catatgtcta
      661 cggacttact atacggttgg ttggaaagaa gcagctcagg cgccctgtac catcttgtag
      721 caacgtaatt ggttatatgg ggttgcacag tggaatgaca cttgaaaaat ccggcatgaa
      781 tacccctagc gagaccaaaa tcgcagatct tcaaacagcc attcaatgtg cacaatatgt
      841 tcccaggctt caaatctcgg tggatgacat ctgcactgtg tatgtacttt agcccacaca
      901 ggatttggta aagaaagtat ttgatatgaa actccgagag ctgcacggac gaatgtatga
      961 cttttgccaa atcataatca atcagttcct ggtagcaata cagtccgtca tagggcgagc
     1021 tggtcactat ctctagatcg attagattta caatattttt atggcccttg aagaagttca
     1081 tgaattttag ctcacggatg gcccttttca aaaggatctc cttattaaag atgttgctga
     1141 tcttctttac cgctatccta gcagctgggc ttcgaccctt aaacttcact gagcacactg
     1201 tcccataggc tcccttgccc aggaattgga ttatctcgta gtatcctggg atggatattc
     1261 tctccttggc aaaaatcgtc ctctgttgag gagcaccgag gttggaggcc acatttatgg
     1321 ctctggtatt atctgtaagt gtgcgattca tatgtatatc tccttcttaa agttaaacaa
     1381 aattatttct agaggggaat tgttatccgc tcacaattcc cctatagtga gtcgtattaa
     1441 tttcgcggga tcgagatcga tctcgatcct ctacgccgga cgcatcgtgg ccggcatcac
     1501 cggcgccaca ggtgcggttg ctggcgccta tatcgccgac atcaccgatg gggaagatcg
     1561 ggctcgccac ttcgggctca tgagcgcttg tttcggcgtg ggtatggtgg caggccccgt
     1621 ggccggggga ctgttgggcg ccatctcctt gcatgcacca ttccttgcgg cggcggtgct
     1681 caacggcctc aacctactac tgggctgctt cctaatgcag gagtcgcata agggagagcg
     1741 tcgagatccc ggacaccatc gaatggcgca aaacctttcg cggtatggca tgatagcgcc
     1801 cggaagagag tcaattcagg gtggtgaatg tgaaaccagt aacgttatac gatgtcgcag
     1861 agtatgccgg tgtctcttat cagaccgttt cccgcgtggt gaaccaggcc agccacgttt
     1921 ctgcgaaaac gcgggaaaaa gtggaagcgg cgatggcgga gctgaattac attcccaacc
     1981 gcgtggcaca acaactggcg ggcaaacagt cgttgctgat tggcgttgcc acctccagtc
     2041 tggccctgca cgcgccgtcg caaattgtcg cggcgattaa atctcgcgcc gatcaactgg
     2101 gtgccagcgt ggtggtgtcg atggtagaac gaagcggcgt cgaagcctgt aaagcggcgg
     2161 tgcacaatct tctcgcgcaa cgcgtcagtg ggctgatcat taactatccg ctggatgacc
     2221 aggatgccat tgctgtggaa gctgcctgca ctaatgttcc ggcgttattt cttgatgtct
     2281 ctgaccagac acccatcaac agtattattt tctcccatga agacggtacg cgactgggcg
     2341 tggagcatct ggtcgcattg ggtcaccagc aaatcgcgct gttagcgggc ccattaagtt
     2401 ctgtctcggc gcgtctgcgt ctggctggct ggcataaata tctcactcgc aatcaaattc
     2461 agccgatagc ggaacgggaa ggcgactgga gtgccatgtc cggttttcaa caaaccatgc
     2521 aaatgctgaa tgagggcatc gttcccactg cgatgctggt tgccaacgat cagatggcgc
     2581 tgggcgcaat gcgcgccatt accgagtccg ggctgcgcgt tggtgcggac atctcggtag
     2641 tgggatacga cgataccgaa gacagctcat gttatatccc gccgttaacc accatcaaac
     2701 aggattttcg cctgctgggg caaaccagcg tggaccgctt gctgcaactc tctcagggcc
     2761 aggcggtgaa gggcaatcag ctgttgcccg tctcactggt gaaaagaaaa accaccctgg
     2821 cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac
     2881 gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt aagttagctc
     2941 actcattagg caccgggatc tcgaccgatg cccttgagag ccttcaaccc agtcagctcc
     3001 ttccggtggg cgcggggcat gactatcgtc gccgcactta tgactgtctt ctttatcatg
     3061 caactcgtag gacaggtgcc ggcagcgctc tgggtcattt tcggcgagga ccgctttcgc
     3121 tggagcgcga cgatgatcgg cctgtcgctt gcggtattcg gaatcttgca cgccctcgct
     3181 caagccttcg tcactggtcc cgccaccaaa cgtttcggcg agaagcaggc cattatcgcc
     3241 ggcatggcgg ccccacgggt gcgcatgatc gtgctcctgt cgttgaggac ccggctaggc
     3301 tggcggggtt gccttactgg ttagcagaat gaatcaccga tacgcgagcg aacgtgaagc
     3361 gactgctgct gcaaaacgtc tgcgacctga gcaacaacat gaatggtctt cggtttccgt
     3421 gtttcgtaaa gtctggaaac gcggaagtca gcgccctgca ccattatgtt ccggatctgc
     3481 atcgcaggat gctgctggct accctgtgga acacctacat ctgtattaac gaagcgctgg
     3541 cattgaccct gagtgatttt tctctggtcc cgccgcatcc ataccgccag ttgtttaccc
     3601 tcacaacgtt ccagtaaccg ggcatgttca tcatcagtaa cccgtatcgt gagcatcctc
     3661 tctcgtttca tcggtatcat tacccccatg aacagaaatc ccccttacac ggaggcatca
     3721 gtgaccaaac aggaaaaaac cgcccttaac atggcccgct ttatcagaag ccagacatta
     3781 acgcttctgg agaaactcaa cgagctggac gcggatgaac aggcagacat ctgtgaatcg
     3841 cttcacgacc acgctgatga gctttaccgc agctgcctcg cgcgtttcgg tgatgacggt
     3901 gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc
     3961 gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggcgcagcc
     4021 atgacccagt cacgtagcga tagcggagtg tatactggct taactatgcg gcatcagagc
     4081 agattgtact gagagtgcac cattgcggtg tgaaataccg cacagatgcg taaggagaaa
     4141 ataccgcatc aggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     4201 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     4261 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     4321 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     4381 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     4441 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     4501 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     4561 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     4621 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     4681 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     4741 gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc
     4801 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     4861 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     4921 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     4981 acgttaaggg attttggtca tgaacaataa aactgtctgc ttacataaac agtaatacaa
     5041 ggggtgttat gagccatatt caacgggaaa cgtcttgctc taggccgcga ttaaattcca
     5101 acatggatgc tgatttatat gggtataaat gggctcgcga taatgtcggg caatcaggtg
     5161 cgacaatcta tcgattgtat gggaagcccg atgcgccaga gttgtttctg aaacatggca
     5221 aaggtagcgt tgccaatgat gttacagatg agatggtcag actaaactgg ctgacggaat
     5281 ttatgcctct tccgaccatc aagcatttta tccgtactcc tgatgatgca tggttactca
     5341 ccactgcgat ccccgggaaa acagcattcc aggtattaga agaatatcct gattcaggtg
     5401 aaaatattgt tgatgcgctg gcagtgttcc tgcgccggtt gcattcgatt cctgtttgta
     5461 attgtccttt taacagcgat cgcgtatttc gtctcgctca ggcgcaatca cgaatgaata
     5521 acggtttggt tgatgcgagt gattttgatg acgagcgtaa tggctggcct gttgaacaag
     5581 tctggaaaga aatgcataaa cttttgccat tctcaccgga ttcagtcgtc actcatggtg
     5641 atttctcact tgataacctt atttttgacg aggggaaatt aataggttgt attgatgttg
     5701 gacgagtcgg aatcgcagac cgataccagg atcttgccat cctatggaac tgcctcggtg
     5761 agttttctcc ttcattacag aaacggcttt ttcaaaaata tggtattgat aatcctgata
     5821 tgaataaatt gcagtttcat ttgatgctcg atgagttttt ctaagaatta attcatgagc
     5881 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     5941 cgaaaagtgc cacctaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt
     6001 gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa
     6061 aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa
     6121 agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac
     6181 gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga
     6241 accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa
     6301 aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc
     6361 tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg tcccattcgc
     6421 ca
//
