LOCUS       sequence-435359-        7218 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  WT SeKaiC for phosphorylation/dephosphorylation study.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7218)
  AUTHORS   Kim YI, Dong G, Carruthers CW Jr, Golden SS, LiWang A
  TITLE     The day/night switch in KaiC, a central oscillator component of the 
            circadian clock of cyanobacteria.
  JOURNAL   Proc Natl Acad Sci U S A. 2008 Sep 2;105(35):12825-30. doi: 
            10.1073/pnas.0800526105. Epub 2008 Aug 26.
  PUBMED    18728181
REFERENCE   2  (bases 1 to 7218)
  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.0800526105"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2008-09-2- 2"; volume: "105"; issue: "35"; pages: "12825-30"
COMMENT     Sequence Label: STREP-SUMO-FLAG-SeKaiC
FEATURES             Location/Qualifiers
     source          1..7218
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     448..467
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     701..718
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    733..872
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(798..817)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     958..980
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(974..1165)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1974..3056)
                     /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     3018..3037
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3057..3134)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3340..3359
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3443..3462
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3443..3461
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3462..3486
                     /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             3501..3523
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3536..3559
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             3569..3586
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3596..3613
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             3626..3919
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             3920..3943
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             5525..5542
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5595..5613)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5609..5656
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      5693..6148
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5780..5799)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5990..6011
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(6240..7055)
                     /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(6240..7055)
                     /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     6963..6982
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     7047..7066
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      join(7177..7218,1..547)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
       61 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
      121 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
      181 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
      241 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
      301 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
      361 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
      421 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
      481 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
      541 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
      601 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
      661 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
      721 agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
      781 caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc
      841 cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg
      901 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
      961 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt
     1021 aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca
     1081 gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa
     1141 gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg
     1201 gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac
     1261 atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc
     1321 agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac
     1381 agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact
     1441 tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg
     1501 tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct
     1561 gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca
     1621 tgcgcacccg tggggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg
     1681 tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg
     1741 acaggccgat catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg
     1801 ctgccggcac ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga
     1861 tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg
     1921 gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc
     1981 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     2041 ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc
     2101 aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg
     2161 gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag
     2221 ctgtcttcgg tatcgtcgta tcccactacc gagatatccg caccaacgcg cagcccggac
     2281 tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg
     2341 ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag
     2401 tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca
     2461 gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg
     2521 tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata
     2581 atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag
     2641 gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg
     2701 acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct
     2761 accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca
     2821 atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt
     2881 ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct
     2941 tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg
     3001 gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc
     3061 accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc
     3121 cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaagca
     3181 gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga
     3241 gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc
     3301 gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc
     3361 gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat
     3421 cgagatctcg atcccgcgaa attaatacga ctcactatag gggaattgtg agcggataac
     3481 aattcccctc tagaaataat tttgtttaac tttaagaagg agatatacca tggaatggag
     3541 ccacccgcag ttcgaaaagg gcagcagcca tcatcatcat catcacagca gcggcctggt
     3601 gccgcgcggc agccatatgg ctagcatgtc ggactcagaa gtcaatcaag aagctaagcc
     3661 agaggtcaag ccagaagtca agcctgagac tcacatcaat ttaaaggtgt ccgatggatc
     3721 ttcagagatc ttcttcaaga tcaaaaagac cactccttta agaaggctga tggaagcgtt
     3781 cgctaaaaga cagggtaagg aaatggactc cttaagattc ttgtacgacg gtattagaat
     3841 ccaagctgat cagacccctg aagatttgga catggaggat aacgatatta ttgaggctca
     3901 cagagaacag attggtggag attacaagga tgacgacgat aagatgactt ccgctgagat
     3961 gactagccct aataataatt ctgagcacca agccatcgct aagatgcgca cgatgattga
     4021 aggctttgat gatattagtc atggcggtct tccaatcggg cgatcgaccc tcgttagtgg
     4081 tacttcagga accggcaaga cccttttttc tattcaattt ctctataacg gtattatcga
     4141 gtttgatgag cctggggttt tcgttacttt cgaagaaacc ccgcaagata tcattaaaaa
     4201 cgcccgtagt tttggctggg atttagccaa gctggtcgat gagggcaaac tatttattct
     4261 tgatgcttca cccgatccag aaggtcaaga ggttgttggc ggcttcgatc tctctgctct
     4321 gattgagcgg attaattatg caattcaaaa gtatcgagcg cggcgggttt caattgactc
     4381 ggtcacgtcc gttttccagc aatatgatgc ctcttctgtg gttcgccgcg aactctttcg
     4441 gttggtagct cgcctaaaac aaattggggc aactacggtc atgaccaccg agcgtatcga
     4501 ggaatatggc ccgatcgctc gttacggtgt tgaggaattt gtctccgata acgtcgtgat
     4561 tctccgcaac gttttggaag gggagcgccg tcgccgcacc ctcgaaatcc tcaagctacg
     4621 tggcaccagc cacatgaaag gggaatatcc gttcacgatt acggatcatg gcatcaatat
     4681 cttcccgctc ggggcaatgc gccttacgca gcgatcgtcg aacgtgcgtg tttcatctgg
     4741 tgtcgtccga ctcgatgaaa tgtgtggtgg gggcttcttt aaggactcaa tcattctggc
     4801 aactggcgct acaggcactg gtaaaactct gttagttagc cgtttcgttg agaatgcttg
     4861 tgctaacaaa gagcgggcga ttctgttcgc ttatgaagag tcacgagctc agctgctccg
     4921 caatgcctat tcatggggaa tggactttga ggagatggag cgccaaaacc tcctcaaaat
     4981 tgtttgcgcc tatcctgaat ctgcaggtct tgaagaccat ttgcagatta ttaagtcgga
     5041 gatcaatgac tttaagccag ctcgtattgc aatcgactcc ctctctgctt tggcgcgggg
     5101 cgttagcaac aatgccttcc gccaatttgt aattggtgtc actggctacg cgaaacaaga
     5161 agaaatcacg ggactattca caaataccag tgatcaattt atgggagcgc attcgattac
     5221 tgactcccat atctcaacaa ttacggatac gattatcttg ctccaatacg tcgagattcg
     5281 tggcgaaatg tcccgcgcca ttaacgtctt caagatgcgc ggatcttggc atgacaaagc
     5341 aatccgcgaa ttcatgatca gcgacaaagg gccggacatc aaggattctt tccggaactt
     5401 tgagcggatt atttcaggtt cgccaacacg gattaccgtc gatgagaaaa gcgaactctc
     5461 gcgaattgtg cgcggcgttc aagaaaaagg gccggagagc tgaaagcttg cggccgcact
     5521 cgagcaccac caccaccacc actgagatcc ggctgctaac aaagcccgaa aggaagctga
     5581 gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt
     5641 cttgaggggt tttttgctga aaggaggaac tatatccgga ttggcgaatg ggacgcgccc
     5701 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     5761 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     5821 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     5881 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc
     5941 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     6001 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt
     6061 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     6121 tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa
     6181 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg aattaattct
     6241 tagaaaaact catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata
     6301 ccatattttt gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat
     6361 aggatggcaa gatcctggta tcggtctgcg attccgactc gtccaacatc aatacaacct
     6421 attaatttcc cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg agtgacgact
     6481 gaatccggtg agaatggcaa aagtttatgc atttctttcc agacttgttc aacaggccag
     6541 ccattacgct cgtcatcaaa atcactcgca tcaaccaaac cgttattcat tcgtgattgc
     6601 gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa
     6661 tgcaaccggc gcaggaacac tgccagcgca tcaacaatat tttcacctga atcaggatat
     6721 tcttctaata cctggaatgc tgttttcccg gggatcgcag tggtgagtaa ccatgcatca
     6781 tcaggagtac ggataaaatg cttgatggtc ggaagaggca taaattccgt cagccagttt
     6841 agtctgacca tctcatctgt aacatcattg gcaacgctac ctttgccatg tttcagaaac
     6901 aactctggcg catcgggctt cccatacaat cgatagattg tcgcacctga ttgcccgaca
     6961 ttatcgcgag cccatttata cccatataaa tcagcatcca tgttggaatt taatcgcggc
     7021 ctagagcaag acgtttcccg ttgaatatgg ctcataacac cccttgtatt actgtttatg
     7081 taagcagaca gttttattgt tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     7141 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     7201 aatctgctgc ttgcaaac
//
