LOCUS       sequence-497720-        6956 bp    DNA     circular SYN 16-JAN-2026
DEFINITION  expresses yeast Isc10 fused to maltose binding protein and Cak1 in 
            bacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6956)
  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 6956)
  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: pZK12
FEATURES             Location/Qualifiers
     source          1..6956
                     /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)."
     CDS             77..1177
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDAALAAAQT"
     CDS             1190..1213
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     promoter        2089..2107
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2108..2132
                     /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             3312..3356
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(3394..3412)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3408..3455
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(3676..4335)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     4250..4269
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(4336..4438)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(4830..4847)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4964..5509)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(5044..5063)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5734..6816)
                     /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     6778..6797
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6817..6894)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        join(6940..6956,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggggaattgt gagcggataa caattcccct gtagaaataa ttttgtttaa ctttaataag
       61 gagatatacc atggcaatga aaatcgaaga aggtaaactg gtaatctgga ttaacggcga
      121 taaaggctat aacggtctcg ctgaagtcgg taagaaattc gagaaagata ccggaattaa
      181 agtcaccgtt gagcatccgg ataaactgga agagaaattc ccacaggttg cggcaactgg
      241 cgatggccct gacattatct tctgggcaca cgaccgcttt ggtggctacg ctcaatctgg
      301 cctgttggct gaaatcaccc cggacaaagc gttccaggac aagctgtatc cgtttacctg
      361 ggatgccgta cgttacaacg gcaagctgat tgcttacccg atcgctgttg aagcgttatc
      421 gctgatttat aacaaagatc tgctgccgaa cccgccaaaa acctgggaag agatcccggc
      481 gctggataaa gaactgaaag cgaaaggtaa gagcgcgctg atgttcaacc tgcaagaacc
      541 gtacttcacc tggccgctga ttgctgctga cgggggttat gcgttcaagt atgaaaacgg
      601 caagtacgac attaaagacg tgggcgtgga taacgctggc gcgaaagcgg gtctgacctt
      661 cctggttgac ctgattaaaa acaaacacat gaatgcagac accgattact ccatcgcaga
      721 agctgccttt aataaaggcg aaacagcgat gaccatcaac ggcccgtggg catggtccaa
      781 catcgacacc agcaaagtga attatggtgt aacggtactg ccgaccttca agggtcaacc
      841 atccaaaccg ttcgttggcg tgctgagcgc aggtattaac gccgccagtc cgaacaaaga
      901 gctggcaaaa gagttcctcg aaaactatct gctgactgat gaaggtctgg aagcggttaa
      961 taaagacaaa ccgctgggtg ccgtagcgct gaagtcttac gaggaagagt tggcgaaaga
     1021 tccacgtatt gccgccacta tggaaaacgc ccagaaaggt gaaatcatgc cgaacatccc
     1081 gcagatgtcc gctttctggt atgccgtgcg tactgcggtg atcaacgccg ccagcggtcg
     1141 tcagactgtc gatgcagccc tggcagccgc gcagactaat gccgccgcac tggaagttct
     1201 gttccagggg cccgctagca tggacgttga cgaaagattg caccaagacg agaaccagac
     1261 ccacccgttt agccagaaaa agagcagcag cttcctgatt aaggagaaag cggctaccaa
     1321 aagcaaggat ctggaacata ttcgtctgcg tgacctgaat tttaaccacc gtaaaaagct
     1381 ggatgacaaa aagctggcga aacaaattcc ggttaaggct aacttcaaaa agccgaatga
     1441 aatcgaagca cgtgcgaatc tgaacagcaa tgaactgacc gacattaacc tggattacat
     1501 cccggacagc ccgagcattg aaaagatcag cggtccggaa gatagcatcg ttgtgacccc
     1561 gcgtaacatt atccatctgc agagcgacag cgatattatc ctggaagaat gcgaacacaa
     1621 ttacgactgt agcccgtttt accgtctgtt caactatgaa aatcgtattg aaccggatga
     1681 ctacgaagct attatcaatg caatcatcac cgatgaaatc gcaggcacct acccggtttt
     1741 tgaacgtgaa ctggaatatc aagaactgaa aagcctggtg cgtaagcgtg attacatcat
     1801 gtactatttt ctgagccgtg actatcgtgg tttctttcag ctgaaagaag aacgtaccct
     1861 gttctaccgt tatccgagca tcgcttacac cagcccgctg cgttatctgg acaacggtag
     1921 cgaaaccgaa caattcaccg gtgacgacga tgaagaactg cagagctttg atttcgaaaa
     1981 caccagcagc gtgcgtaccc tggacagcaa tatttggcgt tgagcggccg cataatgctt
     2041 aagtcgaaca gaaagtaatc gtattgtaca cggccgcata atcgaaatta atacgactca
     2101 ctatagggga attgtgagcg gataacaatt ccccatctta gtatattagt taagtataag
     2161 aaggagatat acatatggca gatctcatga aactggatag tatagacatt acacactgtc
     2221 agttggtcaa atctactaga actgctagga tttataggtc ggatacatat gccattaaat
     2281 gtctagcact agatttcgat atcccgccac ataacgccaa attcgaagta tcgatattaa
     2341 acaaactggg caacaaatgt aagcacatct tacctcttct agagtctaag gctaccgata
     2401 ataatgacct attgttgctg tttccctttg aagagatgaa cctttatgag ttcatgcaaa
     2461 tgcactataa aagagataga agaaaaaaaa atccctatta cgatttgcta aatcccagta
     2521 tcccaattgt tgcggacccc ccagttcaga aatatactaa tcaattggac gtcaatcggt
     2581 attctttgtc ctttttctgg caaatggttg aagggattgc attcttacat gagaacaaga
     2641 tcattcaccg cgacatcaaa ccgcaaaata tcatgctaac gaacaatacc agcaccgtat
     2701 ccccaaagtt gtacataatt gattttggca tctcatatga catggcaaat aactcacaaa
     2761 caagtgcgga acccatggac agcaaggtga cggatataag cacaggaatt tacaaggccc
     2821 cagaagtgct ttttggagta aaatgctatg atggtggcgt ggacgtgtgg tcgttgttga
     2881 taattatttc tcagtggttc cagagagaaa caagccgtat ggggcacgtt ccggccatga
     2941 ttgatgacgg cagcgacgac atgaactcag atggaagcga tttcagactg atttgctcaa
     3001 tattcgaaaa gttgggcata ccgtccattc agaaatggga ggaggttgcg caacacggct
     3061 cggttgatgc atttgttggt atgtttggtg cagatggcga tggtaagtat gtactggacc
     3121 aggagaaaga tgtacagatt agcatcgttg agaggaatat gcctcgactg gacgagattg
     3181 cggatgtcaa agtcaagcag aagttcatta attgtatcct ggggatggtt tcattttcac
     3241 caaacgaaag atggagctgt caaagaatct tgcaagaact agaaaagcca taaggtaccc
     3301 tcgagtctgg taaagaaacc gctgctgcga aatttgaacg ccagcacatg gactcgtcta
     3361 ctagcgcagc ttaattaacc taggctgctg ccaccgctga gcaataacta gcataacccc
     3421 ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa acctcaggca tttgagaagc
     3481 acacggtcac actgcttccg gtagtcaata aaccggtaaa ccagcaatag acataagcgg
     3541 ctatttaacg accctgccct gaaccgacga ccgggtcgaa tttgctttcg aatttctgcc
     3601 attcatccgc ttattatcac ttattcaggc gtagcaacca ggcgtttaag ggcaccaata
     3661 actgccttaa aaaaattacg ccccgccctg ccactcatcg cagtactgtt gtaattcatt
     3721 aagcattctg ccgacatgga agccatcaca aacggcatga tgaacctgaa tcgccagcgg
     3781 catcagcacc ttgtcgcctt gcgtataata tttgcccata gtgaaaacgg gggcgaagaa
     3841 gttgtccata ttggccacgt ttaaatcaaa actggtgaaa ctcacccagg gattggctga
     3901 gacgaaaaac atattctcaa taaacccttt agggaaatag gccaggtttt caccgtaaca
     3961 cgccacatct tgcgaatata tgtgtagaaa ctgccggaaa tcgtcgtggt attcactcca
     4021 gagcgatgaa aacgtttcag tttgctcatg gaaaacggtg taacaagggt gaacactatc
     4081 ccatatcacc agctcaccgt ctttcattgc catacggaac tccggatgag cattcatcag
     4141 gcgggcaaga atgtgaataa aggccggata aaacttgtgc ttatttttct ttacggtctt
     4201 taaaaaggcc gtaatatcca gctgaacggt ctggttatag gtacattgag caactgactg
     4261 aaatgcctca aaatgttctt tacgatgcca ttgggatata tcaacggtgg tatatccagt
     4321 gatttttttc tccattttag cttccttagc tcctgaaaat ctcgataact caaaaaatac
     4381 gcccggtagt gatcttattt cattatggtg aaagttggaa cctcttacgt gccgatcaac
     4441 gtctcatttt cgccaaaagt tggcccaggg cttcccggta tcaacaggga caccaggatt
     4501 tatttattct gcgaagtgat cttccgtcac aggtatttat tcggcgcaaa gtgcgtcggg
     4561 tgatgctgcc aacttactga tttagtgtat gatggtgttt ttgaggtgct ccagtggctt
     4621 ctgtttctat cagctgtccc tcctgttcag ctactgacgg ggtggtgcgt aacggcaaaa
     4681 gcaccgccgg acatcagcgc gagcggagtg tatactggct tactatgttg gcactgatga
     4741 gggtgtcagt gaagtgcttc atgtggcagg agaaaaaagg ctgcaccggt gcgtcagcag
     4801 aatatgtgat acaggatata ttccgcttcc tcgctcactg actcgctacg ctcggtcgtt
     4861 cgactgcggc gagcggaaat ggcttacgaa cggggcggag atttcctgga agatgccagg
     4921 aagatactta acagggaagt gagagggccg cggcaaagcc gtttttccat aggctccgcc
     4981 cccctgacaa gcatcacgaa atctgacgct caaatcagtg gtggcgaaac ccgacaggac
     5041 tataaagata ccaggcgttt ccccctggcg gctccctcgt gcgctctcct gttcctgcct
     5101 ttcggtttac cggtgtcatt ccgctgttat ggccgcgttt gtctcattcc acgcctgaca
     5161 ctcagttccg ggtaggcagt tcgctccaag ctggactgta tgcacgaacc ccccgttcag
     5221 tccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccgga aagacatgca
     5281 aaagcaccac tggcagcagc cactggtaat tgatttagag gagttagtct tgaagtcatg
     5341 cgccggttaa ggctaaactg aaaggacaag ttttggtgac tgcgctcctc caagccagtt
     5401 acctcggttc aaagagttgg tagctcagag aaccttcgaa aaaccgccct gcaaggcggt
     5461 tttttcgttt tcagagcaag agattacgcg cagaccaaaa cgatctcaag aagatcatct
     5521 tattaatcag ataaaatatt tctagatttc agtgcaattt atctcttcaa atgtagcacc
     5581 tgaagtcagc cccatacgat ataagttgta attctcatgt tagtcatgcc ccgcgcccac
     5641 cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa
     5701 tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     5761 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     5821 gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac
     5881 cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa
     5941 atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta
     6001 tcccactacc gagatgtccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc
     6061 gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag
     6121 catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat
     6181 cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga
     6241 gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg
     6301 ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg
     6361 gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc
     6421 atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt
     6481 gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct
     6541 ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag
     6601 ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc
     6661 cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt
     6721 cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc
     6781 atactctgcg acatcgtata acgttactgg tttcacattc accaccctga attgactctc
     6841 ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat
     6901 ctcgacgctc tcccttatgc gactcctgca ttaggaaatt aatacgactc actata
//
