LOCUS       sequence-167902-        5919 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Strain: AM5116, Used for protein purification.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5919)
  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 5919)
  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-KaiB
FEATURES             Location/Qualifiers
     source          1..5919
                     /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(488..781)
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             complement(794..811)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(821..838)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             857..879
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    894..918
                     /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(918..937)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(919..937)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1021..1040)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1246..1323
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1324..2406
                     /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(1343..1362)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3215..3406
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3400..3422)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3508..3650
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3563..3582
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3665..3682)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3836..4424)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3916..3935)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4535..4554)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4546..5361
                     /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             4546..5361
                     /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(4619..4638)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5453..5908)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5590..5611)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5802..5821
                     /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 gcaagctttt
      181 agaagtcgtc ggaatcttga agttcgccgt agagtaaatc aaggccaatc aaaactttct
      241 cacggtcgga taaatcacca ataatccgtc ggacaggcag tggtagaacc ttggcgaggg
      301 ttggcgtcgc taggatttta tcctcttctg ccaactgagg atttttgaga acatcgatca
      361 cctttagagc ataaacacct tgaaattcaa cttcgagaat gttcttgagc gttttgaggg
      421 cacggactga gtttggagta ttgccggcga cgtagagctt gagaatgtag gttttacgag
      481 ggctcattcc accaatctgt tctctgtgag cctcaataat atcgttatcc tccatgtcca
      541 aatcttcagg ggtctgatca gcttggattc taataccgtc gtacaagaat cttaaggagt
      601 ccatttcctt accctgtctt ttagcgaacg cttccatcag ccttcttaaa ggagtggtct
      661 ttttgatctt gaagaagatc tctgaagatc catcggacac ctttaaattg atgtgagtct
      721 caggcttgac ttctggcttg acctctggct tagcttcttg attgacttct gagtccgaca
      781 tgctagccat atggctgccg cgcggcacca ggccgctgct gtgatgatga tgatgatggc
      841 tgctgcccat ggtatatctc cttcttaaag ttaaacaaaa ttatttctag aggggaattg
      901 ttatccgctc acaattcccc tatagtgagt cgtattaatt tcgcgggatc gagatctcga
      961 tcctctacgc cggacgcatc gtggccggca tcaccggcgc cacaggtgcg gttgctggcg
     1021 cctatatcgc cgacatcacc gatggggaag atcgggctcg ccacttcggg ctcatgagcg
     1081 cttgtttcgg cgtgggtatg gtggcaggcc ccgtggccgg gggactgttg ggcgccatct
     1141 ccttgcatgc accattcctt gcggcggcgg tgctcaacgg cctcaaccta ctactgggct
     1201 gcttcctaat gcaggagtcg cataagggag agcgtcgaga tcccggacac catcgaatgg
     1261 cgcaaaacct ttcgcggtat ggcatgatag cgcccggaag agagtcaatt cagggtggtg
     1321 aatgtgaaac cagtaacgtt atacgatgtc gcagagtatg ccggtgtctc ttatcagacc
     1381 gtttcccgcg tggtgaacca ggccagccac gtttctgcga aaacgcggga aaaagtggaa
     1441 gcggcgatgg cggagctgaa ttacattccc aaccgcgtgg cacaacaact ggcgggcaaa
     1501 cagtcgttgc tgattggcgt tgccacctcc agtctggccc tgcacgcgcc gtcgcaaatt
     1561 gtcgcggcga ttaaatctcg cgccgatcaa ctgggtgcca gcgtggtggt gtcgatggta
     1621 gaacgaagcg gcgtcgaagc ctgtaaagcg gcggtgcaca atcttctcgc gcaacgcgtc
     1681 agtgggctga tcattaacta tccgctggat gaccaggatg ccattgctgt ggaagctgcc
     1741 tgcactaatg ttccggcgtt atttcttgat gtctctgacc agacacccat caacagtatt
     1801 attttctccc atgaagacgg tacgcgactg ggcgtggagc atctggtcgc attgggtcac
     1861 cagcaaatcg cgctgttagc gggcccatta agttctgtct cggcgcgtct gcgtctggct
     1921 ggctggcata aatatctcac tcgcaatcaa attcagccga tagcggaacg ggaaggcgac
     1981 tggagtgcca tgtccggttt tcaacaaacc atgcaaatgc tgaatgaggg catcgttccc
     2041 actgcgatgc tggttgccaa cgatcagatg gcgctgggcg caatgcgcgc cattaccgag
     2101 tccgggctgc gcgttggtgc ggatatctcg gtagtgggat acgacgatac cgaagacagc
     2161 tcatgttata tcccgccgtt aaccaccatc aaacaggatt ttcgcctgct ggggcaaacc
     2221 agcgtggacc gcttgctgca actctctcag ggccaggcgg tgaagggcaa tcagctgttg
     2281 cccgtctcac tggtgaaaag aaaaaccacc ctggcgccca atacgcaaac cgcctctccc
     2341 cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg
     2401 cagtgagcgc aacgcaatta atgtaagtta gctcactcat taggcaccgg gatctcgacc
     2461 gatgcccttg agagccttca acccagtcag ctccttccgg tgggcgcggg gcatgactat
     2521 cgtcgccgca cttatgactg tcttctttat catgcaactc gtaggacagg tgccggcagc
     2581 gctctgggtc attttcggcg aggaccgctt tcgctggagc gcgacgatga tcggcctgtc
     2641 gcttgcggta ttcggaatct tgcacgccct cgctcaagcc ttcgtcactg gtcccgccac
     2701 caaacgtttc ggcgagaagc aggccattat cgccggcatg gcggccccac gggtgcgcat
     2761 gatcgtgctc ctgtcgttga ggacccggct aggctggcgg ggttgcctta ctggttagca
     2821 gaatgaatca ccgatacgcg agcgaacgtg aagcgactgc tgctgcaaaa cgtctgcgac
     2881 ctgagcaaca acatgaatgg tcttcggttt ccgtgtttcg taaagtctgg aaacgcggaa
     2941 gtcagcgccc tgcaccatta tgttccggat ctgcatcgca ggatgctgct ggctaccctg
     3001 tggaacacct acatctgtat taacgaagcg ctggcattga ccctgagtga tttttctctg
     3061 gtcccgccgc atccataccg ccagttgttt accctcacaa cgttccagta accgggcatg
     3121 ttcatcatca gtaacccgta tcgtgagcat cctctctcgt ttcatcggta tcattacccc
     3181 catgaacaga aatccccctt acacggaggc atcagtgacc aaacaggaaa aaaccgccct
     3241 taacatggcc cgctttatca gaagccagac attaacgctt ctggagaaac tcaacgagct
     3301 ggacgcggat gaacaggcag acatctgtga atcgcttcac gaccacgctg atgagcttta
     3361 ccgcagctgc ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc
     3421 ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc
     3481 gtcagcgggt gttggcgggt gtcggggcgc agccatgacc cagtcacgta gcgatagcgg
     3541 agtgtatact ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccatata
     3601 tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg cgctcttccg
     3661 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     3721 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     3781 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     3841 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     3901 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     3961 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     4021 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     4081 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     4141 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     4201 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     4261 acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     4321 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     4381 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     4441 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     4501 acaataaaac tgtctgctta cataaacagt aatacaaggg gtgttatgag ccatattcaa
     4561 cgggaaacgt cttgctctag gccgcgatta aattccaaca tggatgctga tttatatggg
     4621 tataaatggg ctcgcgataa tgtcgggcaa tcaggtgcga caatctatcg attgtatggg
     4681 aagcccgatg cgccagagtt gtttctgaaa catggcaaag gtagcgttgc caatgatgtt
     4741 acagatgaga tggtcagact aaactggctg acggaattta tgcctcttcc gaccatcaag
     4801 cattttatcc gtactcctga tgatgcatgg ttactcacca ctgcgatccc cgggaaaaca
     4861 gcattccagg tattagaaga atatcctgat tcaggtgaaa atattgttga tgcgctggca
     4921 gtgttcctgc gccggttgca ttcgattcct gtttgtaatt gtccttttaa cagcgatcgc
     4981 gtatttcgtc tcgctcaggc gcaatcacga atgaataacg gtttggttga tgcgagtgat
     5041 tttgatgacg agcgtaatgg ctggcctgtt gaacaagtct ggaaagaaat gcataaactt
     5101 ttgccattct caccggattc agtcgtcact catggtgatt tctcacttga taaccttatt
     5161 tttgacgagg ggaaattaat aggttgtatt gatgttggac gagtcggaat cgcagaccga
     5221 taccaggatc ttgccatcct atggaactgc ctcggtgagt tttctccttc attacagaaa
     5281 cggctttttc aaaaatatgg tattgataat cctgatatga ataaattgca gtttcatttg
     5341 atgctcgatg agtttttcta agaattaatt catgagcgga tacatatttg aatgtattta
     5401 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctaaattgta
     5461 agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac
     5521 caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg
     5581 agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa
     5641 gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt
     5701 tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt
     5761 agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga
     5821 gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc
     5881 gcgcttaatg cgccgctaca gggcgcgtcc cattcgcca
//
