LOCUS       sequence-167934-        6465 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Strain: AM5115, Used for protein purification.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6465)
  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 6465)
  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: pET28-SUMO-KaiA
FEATURES             Location/Qualifiers
     source          1..6465
                     /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"
     CDS             complement(1034..1327)
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             complement(1340..1357)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(1367..1384)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1403..1425
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1440..1464
                     /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(1464..1483)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1465..1483)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1567..1586)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1792..1869
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1870..2952
                     /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(1889..1908)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3761..3952
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3946..3968)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4054..4196
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4109..4128
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4211..4228)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4382..4970)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4462..4481)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5081..5100)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5092..5907
                     /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             5092..5907
                     /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(5165..5184)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5999..6454)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6136..6157)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6348..6367
                     /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 gcaagctttc
      181 aggtttctcg tgggatagac cgtcgataca tctcacaaag atgtgcaatt acatcaatta
      241 aagtcagccg ataatccagc aaaatgtcct ctgaacgtcc ctctacgcgg agcttcttgg
      301 caaactcgtc catcagctcc atgtgaattt ctaccacttt ggtgactgga acatcggcaa
      361 agaaagccat gttgacgaag ttgtcaatgc tctggttgag gttgctattc ggcgaaaaat
      421 agctcaaaac gatttcacga tagctagtct gcatcgcttg gtgcagcttt tggctttcgt
      481 aggcgggtag gttgcgcaga aagcgatcgg gatcacgctt gtagtagacc ccgagatagc
      541 ctaggcgctc ttgtagtcgc tgagcgaggt cccgctgctg gctcgatagc tcgggatcgt
      601 ggttggcccc catcagcatg atgtggtcgg ccatggtctc gaccggggct aagcgcagaa
      661 attcagccag tgcagcatca acttggtagg gcaattgctc gagctgatgg ataccgaggt
      721 gcagttcagc gctgtgatag agctgttctt tggctggttc atcgggatcc tcactgtcgc
      781 gatcgcctac gacaatcgct ggtaccacca ctccctcaaa gcagagctgc tgaacaactg
      841 ccctgaagct gggattggct gccactaaaa tcaggcagtc gatttggtca cgatgggttt
      901 gggcatactc caagagcatt tcgccagact cacagacttg gagttgatag cgatcggccg
      961 acagcgcccg ctggcaatcc tgcaaaattg ccgtcgattc cacccaaatg cagattgcaa
     1021 tttgcgagag cactccacca atctgttctc tgtgagcctc aataatatcg ttatcctcca
     1081 tgtccaaatc ttcaggggtc tgatcagctt ggattctaat accgtcgtac aagaatctta
     1141 aggagtccat ttccttaccc tgtcttttag cgaacgcttc catcagcctt cttaaaggag
     1201 tggtcttttt gatcttgaag aagatctctg aagatccatc ggacaccttt aaattgatgt
     1261 gagtctcagg cttgacttct ggcttgacct ctggcttagc ttcttgattg acttctgagt
     1321 ccgacatgct agccatatgg ctgccgcgcg gcaccaggcc gctgctgtga tgatgatgat
     1381 gatggctgct gcccatggta tatctccttc ttaaagttaa acaaaattat ttctagaggg
     1441 gaattgttat ccgctcacaa ttcccctata gtgagtcgta ttaatttcgc gggatcgaga
     1501 tctcgatcct ctacgccgga cgcatcgtgg ccggcatcac cggcgccaca ggtgcggttg
     1561 ctggcgccta tatcgccgac atcaccgatg gggaagatcg ggctcgccac ttcgggctca
     1621 tgagcgcttg tttcggcgtg ggtatggtgg caggccccgt ggccggggga ctgttgggcg
     1681 ccatctcctt gcatgcacca ttccttgcgg cggcggtgct caacggcctc aacctactac
     1741 tgggctgctt cctaatgcag gagtcgcata agggagagcg tcgagatccc ggacaccatc
     1801 gaatggcgca aaacctttcg cggtatggca tgatagcgcc cggaagagag tcaattcagg
     1861 gtggtgaatg tgaaaccagt aacgttatac gatgtcgcag agtatgccgg tgtctcttat
     1921 cagaccgttt cccgcgtggt gaaccaggcc agccacgttt ctgcgaaaac gcgggaaaaa
     1981 gtggaagcgg cgatggcgga gctgaattac attcccaacc gcgtggcaca acaactggcg
     2041 ggcaaacagt cgttgctgat tggcgttgcc acctccagtc tggccctgca cgcgccgtcg
     2101 caaattgtcg cggcgattaa atctcgcgcc gatcaactgg gtgccagcgt ggtggtgtcg
     2161 atggtagaac gaagcggcgt cgaagcctgt aaagcggcgg tgcacaatct tctcgcgcaa
     2221 cgcgtcagtg ggctgatcat taactatccg ctggatgacc aggatgccat tgctgtggaa
     2281 gctgcctgca ctaatgttcc ggcgttattt cttgatgtct ctgaccagac acccatcaac
     2341 agtattattt tctcccatga agacggtacg cgactgggcg tggagcatct ggtcgcattg
     2401 ggtcaccagc aaatcgcgct gttagcgggc ccattaagtt ctgtctcggc gcgtctgcgt
     2461 ctggctggct ggcataaata tctcactcgc aatcaaattc agccgatagc ggaacgggaa
     2521 ggcgactgga gtgccatgtc cggttttcaa caaaccatgc aaatgctgaa tgagggcatc
     2581 gttcccactg cgatgctggt tgccaacgat cagatggcgc tgggcgcaat gcgcgccatt
     2641 accgagtccg ggctgcgcgt tggtgcggat atctcggtag tgggatacga cgataccgaa
     2701 gacagctcat gttatatccc gccgttaacc accatcaaac aggattttcg cctgctgggg
     2761 caaaccagcg tggaccgctt gctgcaactc tctcagggcc aggcggtgaa gggcaatcag
     2821 ctgttgcccg tctcactggt gaaaagaaaa accaccctgg cgcccaatac gcaaaccgcc
     2881 tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa
     2941 agcgggcagt gagcgcaacg caattaatgt aagttagctc actcattagg caccgggatc
     3001 tcgaccgatg cccttgagag ccttcaaccc agtcagctcc ttccggtggg cgcggggcat
     3061 gactatcgtc gccgcactta tgactgtctt ctttatcatg caactcgtag gacaggtgcc
     3121 ggcagcgctc tgggtcattt tcggcgagga ccgctttcgc tggagcgcga cgatgatcgg
     3181 cctgtcgctt gcggtattcg gaatcttgca cgccctcgct caagccttcg tcactggtcc
     3241 cgccaccaaa cgtttcggcg agaagcaggc cattatcgcc ggcatggcgg ccccacgggt
     3301 gcgcatgatc gtgctcctgt cgttgaggac ccggctaggc tggcggggtt gccttactgg
     3361 ttagcagaat gaatcaccga tacgcgagcg aacgtgaagc gactgctgct gcaaaacgtc
     3421 tgcgacctga gcaacaacat gaatggtctt cggtttccgt gtttcgtaaa gtctggaaac
     3481 gcggaagtca gcgccctgca ccattatgtt ccggatctgc atcgcaggat gctgctggct
     3541 accctgtgga acacctacat ctgtattaac gaagcgctgg cattgaccct gagtgatttt
     3601 tctctggtcc cgccgcatcc ataccgccag ttgtttaccc tcacaacgtt ccagtaaccg
     3661 ggcatgttca tcatcagtaa cccgtatcgt gagcatcctc tctcgtttca tcggtatcat
     3721 tacccccatg aacagaaatc ccccttacac ggaggcatca gtgaccaaac aggaaaaaac
     3781 cgcccttaac atggcccgct ttatcagaag ccagacatta acgcttctgg agaaactcaa
     3841 cgagctggac gcggatgaac aggcagacat ctgtgaatcg cttcacgacc acgctgatga
     3901 gctttaccgc agctgcctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca
     3961 gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca
     4021 gggcgcgtca gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga
     4081 tagcggagtg tatactggct taactatgcg gcatcagagc agattgtact gagagtgcac
     4141 catatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgct
     4201 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     4261 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     4321 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     4381 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     4441 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     4501 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     4561 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     4621 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     4681 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     4741 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     4801 ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta
     4861 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     4921 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     4981 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     5041 tcatgaacaa taaaactgtc tgcttacata aacagtaata caaggggtgt tatgagccat
     5101 attcaacggg aaacgtcttg ctctaggccg cgattaaatt ccaacatgga tgctgattta
     5161 tatgggtata aatgggctcg cgataatgtc gggcaatcag gtgcgacaat ctatcgattg
     5221 tatgggaagc ccgatgcgcc agagttgttt ctgaaacatg gcaaaggtag cgttgccaat
     5281 gatgttacag atgagatggt cagactaaac tggctgacgg aatttatgcc tcttccgacc
     5341 atcaagcatt ttatccgtac tcctgatgat gcatggttac tcaccactgc gatccccggg
     5401 aaaacagcat tccaggtatt agaagaatat cctgattcag gtgaaaatat tgttgatgcg
     5461 ctggcagtgt tcctgcgccg gttgcattcg attcctgttt gtaattgtcc ttttaacagc
     5521 gatcgcgtat ttcgtctcgc tcaggcgcaa tcacgaatga ataacggttt ggttgatgcg
     5581 agtgattttg atgacgagcg taatggctgg cctgttgaac aagtctggaa agaaatgcat
     5641 aaacttttgc cattctcacc ggattcagtc gtcactcatg gtgatttctc acttgataac
     5701 cttatttttg acgaggggaa attaataggt tgtattgatg ttggacgagt cggaatcgca
     5761 gaccgatacc aggatcttgc catcctatgg aactgcctcg gtgagttttc tccttcatta
     5821 cagaaacggc tttttcaaaa atatggtatt gataatcctg atatgaataa attgcagttt
     5881 catttgatgc tcgatgagtt tttctaagaa ttaattcatg agcggataca tatttgaatg
     5941 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctaa
     6001 attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt
     6061 tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata
     6121 gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac
     6181 gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa
     6241 tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc
     6301 cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg
     6361 aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca
     6421 cccgccgcgc ttaatgcgcc gctacagggc gcgtcccatt cgcca
//
