LOCUS       sequence-167953-        7455 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Strain: AM5117, Used for protein purification.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7455)
  AUTHORS   Kim YI, Vinyard DJ, Ananyev GM, Dismukes GC, Golden SS
  TITLE     Oxidized quinones signal onset of darkness directly to the 
            cyanobacterial circadian oscillator.
  JOURNAL   Proc Natl Acad Sci U S A. 2012 Oct 30;109(44):17765-9. doi: 
            10.1073/pnas.1216401109. Epub 2012 Oct 15.
  PUBMED    23071342
REFERENCE   2  (bases 1 to 7455)
  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: 
            "10.1073/pnas.1216401109"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2012-10-30- 30"; volume: "109"; issue: "44"; pages: "17765-9"
COMMENT     Sequence Label: pET41-GST-KaiC
FEATURES             Location/Qualifiers
     source          1..7455
                     /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(150..173)
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             complement(1751..1774)
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             complement(1787..1801)
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
     CDS             complement(1832..1876)
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             complement(1892..1909)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(1919..1936)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(1964..2617)
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     complement(1984..2006)
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     RBS             2625..2647
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    2662..2686
                     /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(2686..2705)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2687..2705)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2793..2812)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        3018..3095
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3096..4178
                     /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(3115..3134)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4750..4941
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4935..4957)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    5043..5185
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     5098..5117
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5200..5217)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5371..5959)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5451..5470)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(6070..6089)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             6081..6896
                     /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             6081..6896
                     /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(6154..6173)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(6989..7444)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7126..7147)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7338..7357
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgtttagcag cctaggtatt aatcaattag tggtggtggt ggtggtggtg gtgctcgagt
      181 actagctctc cggccctttt tcttgaacgc cgcgcacaat tcgcgagagt tcgcttttct
      241 catcgacggt aatccgtgtt ggcgaacctg aaataatccg ctcaaagttc cggaaagaat
      301 ccttgatgtc cggccctttg tcgctgatca tgaattcgcg gattgctttg tcatgccaag
      361 atccgcgcat cttgaagacg ttaatggcgc gggacatttc gccacgaatc tcgacgtatt
      421 ggagcaagat aatcgtatcc gtaattgttg agatatggga gtcagtaatc gaatgcgctc
      481 ccataaattg atcactggta tttgtgaata gtcccgtgat ttcttcttgt ttcgcgtagc
      541 cagtgacacc aattacaaat tggcggaagg cattgttgct aacgccccgc gccaaagcag
      601 agagggagtc gattgcaata cgagctggct taaagtcatt gatctccgac ttaataatct
      661 gcaaatggtc ttcaagacct gcagattcag gataggcgca aacaattttg aggaggtttt
      721 ggcgctccat ctcctcaaag tccattcccc atgaataggc attgcggagc agctgagctc
      781 gtgactcttc ataagcgaac agaatcgccc gctctttgtt agcacaagca ttctcaacga
      841 aacggctaac taacagagtt ttaccagtgc ctgtagcgcc agttgccaga atgattgagt
      901 ccttaaagaa gcccccacca cacatttcat cgagtcggac gacaccagat gaaacacgca
      961 cgttcgacga tcgctgcgta aggcgcattg ccccgagcgg gaagatattg atgccatgat
     1021 ccgtaatcgt gaacggatat tcccctttca tgtggctggt gccacgtagc ttgaggattt
     1081 cgagggtgcg gcgacggcgc tccccttcca aaacgttgcg gagaatcacg acgttatcgg
     1141 agacaaattc ctcaacaccg taacgagcga tcgggccata ttcctcgata cgctcggtgg
     1201 tcatgaccgt agttgcccca atttgtttta ggcgagctac caaccgaaag agttcgcggc
     1261 gaaccacaga agaggcatca tattgctgga aaacggacgt gaccgagtca attgaaaccc
     1321 gccgcgctcg atacttttga attgcataat taatccgctc aatcagagca gagagatcga
     1381 agccgccaac aacctcttga ccttctggat cgggtgaagc atcaagaata aatagtttgc
     1441 cctcatcgac cagcttggct aaatcccagc caaaactacg ggcgttttta atgatatctt
     1501 gcggggtttc ttcgaaagta acgaaaaccc caggctcatc aaactcgata ataccgttat
     1561 agagaaattg aatagaaaaa agggtcttgc cggttcctga agtaccacta acgagggtcg
     1621 atcgcccgat tggaagaccg ccatgactaa tatcatcaaa gccttcaatc atcgtgcgca
     1681 tcttagcgat ggcttggtgc tcagaattat tattagggct agtcatctca gcggaagtca
     1741 tggatcccag gggcccctgg aacagaactt ccagagccat gggactcttg tcgtcgtcat
     1801 caccggagcc accaccggta cccagatctg ggctgtccat gtgctggcgt tcgaatttag
     1861 cagcagcggt ttctttcata ccaattgcag tactaccgcg tggcaccaga cccgcggagt
     1921 gatggtgatg gtgatgacca gaaccactag ttgaaccatc cgattttgga ggatggtcgc
     1981 caccaccaaa cgtggcttgc cagccctgca aaggccatgc tatatacttg ctggatttca
     2041 agtacttatc aatttgtggg atagcttcaa tacgtttttt aaaacaaact aattttggga
     2101 acgcatccag gcacattggg tccatgtata aaacaacatc aagagcgtca tacaacatga
     2161 agtcaggatg ggttacatga tcaccattta aatatgtttt atgacataaa cgatcttcga
     2221 acattttcag catttcaggt agcttgctaa gaaaatcaac tttgagagtt tcaaagtctt
     2281 tactatatgc aattctcgaa acaccgtatc taatatccaa aaccgctcct tcaagcattg
     2341 aaatctctgc acgctctttt ggacaaccac ccaacatgtt gtgcttgtca gctatataac
     2401 gtatgatggc catagactgt gttaatttaa catcaccatc aatataataa ggaagattgg
     2461 gaaactccaa acccaattca aactttttgt ttcgccattt atcaccttca tcgcgctcat
     2521 acaaatgctc ttcatatttt tcttcaagat attccaaaag aagtcgagtg ggttgcacaa
     2581 ggcccttaat tttccaataa cctagtatag gggacatatg tatatctcct tcttaaagtt
     2641 aaacaaaatt atttctagag gggaattgtt atccgctcac aattccccta tagtgagtcg
     2701 tattaatttc gcgggatcga gatcgatctc gatcctctac gccggacgca tcgtggccgg
     2761 catcaccggc gccacaggtg cggttgctgg cgcctatatc gccgacatca ccgatgggga
     2821 agatcgggct cgccacttcg ggctcatgag cgcttgtttc ggcgtgggta tggtggcagg
     2881 ccccgtggcc gggggactgt tgggcgccat ctccttgcat gcaccattcc ttgcggcggc
     2941 ggtgctcaac ggcctcaacc tactactggg ctgcttccta atgcaggagt cgcataaggg
     3001 agagcgtcga gatcccggac accatcgaat ggcgcaaaac ctttcgcggt atggcatgat
     3061 agcgcccgga agagagtcaa ttcagggtgg tgaatgtgaa accagtaacg ttatacgatg
     3121 tcgcagagta tgccggtgtc tcttatcaga ccgtttcccg cgtggtgaac caggccagcc
     3181 acgtttctgc gaaaacgcgg gaaaaagtgg aagcggcgat ggcggagctg aattacattc
     3241 ccaaccgcgt ggcacaacaa ctggcgggca aacagtcgtt gctgattggc gttgccacct
     3301 ccagtctggc cctgcacgcg ccgtcgcaaa ttgtcgcggc gattaaatct cgcgccgatc
     3361 aactgggtgc cagcgtggtg gtgtcgatgg tagaacgaag cggcgtcgaa gcctgtaaag
     3421 cggcggtgca caatcttctc gcgcaacgcg tcagtgggct gatcattaac tatccgctgg
     3481 atgaccagga tgccattgct gtggaagctg cctgcactaa tgttccggcg ttatttcttg
     3541 atgtctctga ccagacaccc atcaacagta ttattttctc ccatgaagac ggtacgcgac
     3601 tgggcgtgga gcatctggtc gcattgggtc accagcaaat cgcgctgtta gcgggcccat
     3661 taagttctgt ctcggcgcgt ctgcgtctgg ctggctggca taaatatctc actcgcaatc
     3721 aaattcagcc gatagcggaa cgggaaggcg actggagtgc catgtccggt tttcaacaaa
     3781 ccatgcaaat gctgaatgag ggcatcgttc ccactgcgat gctggttgcc aacgatcaga
     3841 tggcgctggg cgcaatgcgc gccattaccg agtccgggct gcgcgttggt gcggacatct
     3901 cggtagtggg atacgacgat accgaagaca gctcatgtta tatcccgccg ttaaccacca
     3961 tcaaacagga ttttcgcctg ctggggcaaa ccagcgtgga ccgcttgctg caactctctc
     4021 agggccaggc ggtgaagggc aatcagctgt tgcccgtctc actggtgaaa agaaaaacca
     4081 ccctggcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc
     4141 tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtaagt
     4201 tagctcactc attaggcacc gggatctcga ccgatgccct tgagagcctt caacccagtc
     4261 agctccttcc ggtgggcgcg gggcatgact agcatgatcg tgctcctgtc gttgaggacc
     4321 cggctaggct ggcggggttg ccttactggt tagcagaatg aatcaccgat acgcgagcga
     4381 acgtgaagcg actgctgctg caaaacgtct gcgacctgag caacaacatg aatggtcttc
     4441 ggtttccgtg tttcgtaaag tctggaaacg cggaagtcag cgccctgcac cattatgttc
     4501 cggatctgca tcgcaggatg ctgctggcta ccctgtggaa cacctacatc tgtattaacg
     4561 aagcgctggc attgaccctg agtgattttt ctctggtccc gccgcatcca taccgccagt
     4621 tgtttaccct cacaacgttc cagtaaccgg gcatgttcat catcagtaac ccgtatcgtg
     4681 agcatcctct ctcgtttcat cggtatcatt acccccatga acagaaatcc cccttacacg
     4741 gaggcatcag tgaccaaaca ggaaaaaacc gcccttaaca tggcccgctt tatcagaagc
     4801 cagacattaa cgcttctgga gaaactcaac gagctggacg cggatgaaca ggcagacatc
     4861 tgtgaatcgc ttcacgacca cgctgatgag ctttaccgca gctgcctcgc gcgtttcggt
     4921 gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa
     4981 gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg
     5041 ggcgcagcca tgacccagtc acgtagcgat agcggagtgt atactggctt aactatgcgg
     5101 catcagagca gattgtactg agagtgcacc atatatgcgg tgtgaaatac cgcacagatg
     5161 cgtaaggaga aaataccgca tcaggcgctc ttccgcttcc tcgctcactg actcgctgcg
     5221 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     5281 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     5341 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     5401 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     5461 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     5521 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     5581 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     5641 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     5701 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     5761 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt
     5821 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     5881 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     5941 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     6001 gaacgaaaac tcacgttaag ggattttggt catgaacaat aaaactgtct gcttacataa
     6061 acagtaatac aaggggtgtt atgagccata ttcaacggga aacgtcttgc tctaggccgc
     6121 gattaaattc caacatggat gctgatttat atgggtataa atgggctcgc gataatgtcg
     6181 ggcaatcagg tgcgacaatc tatcgattgt atgggaagcc cgatgcgcca gagttgtttc
     6241 tgaaacatgg caaaggtagc gttgccaatg atgttacaga tgagatggtc agactaaact
     6301 ggctgacgga atttatgcct cttccgacca tcaagcattt tatccgtact cctgatgatg
     6361 catggttact caccactgcg atccccggga aaacagcatt ccaggtatta gaagaatatc
     6421 ctgattcagg tgaaaatatt gttgatgcgc tggcagtgtt cctgcgccgg ttgcattcga
     6481 ttcctgtttg taattgtcct tttaacagcg atcgcgtatt tcgtctcgct caggcgcaat
     6541 cacgaatgaa taacggtttg gttgatgcga gtgattttga tgacgagcgt aatggctggc
     6601 ctgttgaaca agtctggaaa gaaatgcata aacttttgcc attctcaccg gattcagtcg
     6661 tcactcatgg tgatttctca cttgataacc ttatttttga cgaggggaaa ttaataggtt
     6721 gtattgatgt tggacgagtc ggaatcgcag accgatacca ggatcttgcc atcctatgga
     6781 actgcctcgg tgagttttct ccttcattac agaaacggct ttttcaaaaa tatggtattg
     6841 ataatcctga tatgaataaa ttgcagtttc atttgatgct cgatgagttt ttctaagaat
     6901 taattcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     6961 cgcacatttc cccgaaaagt gccacctgaa attgtaaacg ttaatatttt gttaaaattc
     7021 gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc
     7081 ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag
     7141 agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc
     7201 gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa
     7261 gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg
     7321 aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt
     7381 gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc
     7441 gcgtcccatt cgcca
//
