LOCUS       sequence-457223-        7188 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For expression of T. elongatus KaiC protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7188)
  TITLE     LiWang lab plasmids
REFERENCE   2  (bases 1 to 7188)
  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"
COMMENT     Sequence Label: pET28b-SUMO-FLAG-TeKaiC
FEATURES             Location/Qualifiers
     source          1..7188
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(23..70)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     66..84
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(137..154)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(1733..1756)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(1757..2050)
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             complement(2063..2080)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(2090..2107)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             2126..2148
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    2163..2187
                     /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(2187..2206)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2188..2206)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2290..2309)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2515..2592
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2593..3675
                     /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(2612..2631)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4484..4675
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4669..4691)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4777..4916
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4832..4851
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4931..4948)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5102..5690)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5182..5201)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5801..5820)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5812..6627
                     /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             5812..6627
                     /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(5885..5904)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(6719..7174)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6856..6877)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7068..7087
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 cggatatagt tcctcctttc agcaaaaaac ccctcaagac ccgtttagag gccccaaggg
       61 gttatgctag ttattgctca gcggtggcag cagccaactc agcttccttt cgggctttgt
      121 tagcagccgg atctcagtgg tggtggtggt ggtgctcgag tgcggccgca agctttcact
      181 cgctctctag atcctgcatc cccttggcaa ttcgcgccag ctctgttttt tcgtccacgg
      241 aaatgcgggt gggggtaccg ctaataatcc cctcaaagtt gcggaaggaa tcgcggattt
      301 ctgccccctt ctcagtgatc acatactccc gaatcccctt gtcgtgccaa gagccacgca
      361 tcttaaagac attaattgcc cgcgacatct caccgcggat ttccacgtac tgcaacagca
      421 aaatggtgtc tgtaattgtg gagatatggg actcggtaat cgagttggac cccataaact
      481 gatccgtcgt gttggtgaaa aagccagtga tttcctcctg tttggcaaat ccagtaaccc
      541 cgattacaaa ctgccggaag gcattgttac tcaccccccg cgccaacgca gacaaagagt
      601 caatcgccac ccgtgagggc ttaaagtccg caatctccga cttgataatt tgcaggtgat
      661 cctcaagccc cgctgactct ggataggcac aaataatccg caacaaaccg cgccgttcta
      721 actcctcaaa atcaataccc caagaggagg cattgcgcga caactgcgcc cgcgattctt
      781 cataggcaaa cagcagggct cgttctccct gttggcagcc cgtctccaag aatttactga
      841 ccaagagcgt cttgccagta cccgtagcgc ccgtggccaa aataattgaa tccttgaaga
      901 agccaccgcc acacatctcg tcgagggtct tgacccctga agacacccgc acattcgatg
      961 agcgctgagt caagcgcatg gcccccaacg ggaagatgtt aataccattg ttgatcgtaa
     1021 agggatattc ccccttcatg tgggtggtgc cccgcagctt gagaatttcg accgtgcgcc
     1081 gccgcctttc tccctcgaga acattccgca aaatgaccac attgtcggag acaaactcct
     1141 caacaccaaa acgcgccaca gggccgtatt catctacccg ctcagtggtc ataatcgtgg
     1201 tcacgcccag ttgcttgagg cgaaaagcca agcgaaaaat ttcccgccgc accacggagg
     1261 ccgcatcgta ttgctggaac actgctgtga ccgaatcaat ggagacccgg gttgctttgt
     1321 atttgcgaat ggcatactga atgcgctcaa tcagagcaga taagtcaaag tcaccagcca
     1381 cctcttggcc atcgggatcc ggagaagcat ccaggataaa tagcttgcct tgatcaatca
     1441 gactttgcag gttccagcca aaactgaggg cgtttttgat aatatcttgg ggggattctt
     1501 caaatgtaac aaatatacct ggctcattaa aaatggtaat gccattgtag agaaactgaa
     1561 ctgcaaaaag ggtcttccct gtgcctgaag tgccgctgac taaggtggtg cgtccttggg
     1621 gaagtccccc atgactgata tcgtcaaagc cctcaatcat cgtcgggatt ttcttgactt
     1681 ccgcagagga ctgctccgtt ggactagact gatgttccgg taggtttgtc atcttatcgt
     1741 cgtcatcctt gtaatctcca ccaatctgtt ctctgtgagc ctcaataata tcgttatcct
     1801 ccatgtccaa atcttcaggg gtctgatcag cttggattct aataccgtcg tacaagaatc
     1861 ttaaggagtc catttcctta ccctgtcttt tagcgaacgc ttccatcagc cttcttaaag
     1921 gagtggtctt tttgatcttg aagaagatct ctgaagatcc atcggacacc tttaaattga
     1981 tgtgagtctc aggcttgact tctggcttga cctctggctt agcttcttga ttgacttctg
     2041 agtccgacat gctagccata tggctgccgc gcggcaccag gccgctgctg tgatgatgat
     2101 gatgatggct gctgcccatg gtatatctcc ttcttaaagt taaacaaaat tatttctaga
     2161 ggggaattgt tatccgctca caattcccct atagtgagtc gtattaattt cgcgggatcg
     2221 agatctcgat cctctacgcc ggacgcatcg tggccggcat caccggcgcc acaggtgcgg
     2281 ttgctggcgc ctatatcgcc gacatcaccg atggggaaga tcgggctcgc cacttcgggc
     2341 tcatgagcgc ttgtttcggc gtgggtatgg tggcaggccc cgtggccggg ggactgttgg
     2401 gcgccatctc cttgcatgca ccattccttg cggcggcggt gctcaacggc ctcaacctac
     2461 tactgggctg cttcctaatg caggagtcgc ataagggaga gcgtcgagat cccggacacc
     2521 atcgaatggc gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga gagtcaattc
     2581 agggtggtga atgtgaaacc agtaacgtta tacgatgtcg cagagtatgc cggtgtctct
     2641 tatcagaccg tttcccgcgt ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa
     2701 aaagtggaag cggcgatggc ggagctgaat tacattccca accgcgtggc acaacaactg
     2761 gcgggcaaac agtcgttgct gattggcgtt gccacctcca gtctggccct gcacgcgccg
     2821 tcgcaaattg tcgcggcgat taaatctcgc gccgatcaac tgggtgccag cgtggtggtg
     2881 tcgatggtag aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg
     2941 caacgcgtca gtgggctgat cattaactat ccgctggatg accaggatgc cattgctgtg
     3001 gaagctgcct gcactaatgt tccggcgtta tttcttgatg tctctgacca gacacccatc
     3061 aacagtatta ttttctccca tgaagacggt acgcgactgg gcgtggagca tctggtcgca
     3121 ttgggtcacc agcaaatcgc gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg
     3181 cgtctggctg gctggcataa atatctcact cgcaatcaaa ttcagccgat agcggaacgg
     3241 gaaggcgact ggagtgccat gtccggtttt caacaaacca tgcaaatgct gaatgagggc
     3301 atcgttccca ctgcgatgct ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc
     3361 attaccgagt ccgggctgcg cgttggtgcg gatatctcgg tagtgggata cgacgatacc
     3421 gaagacagct catgttatat cccgccgtta accaccatca aacaggattt tcgcctgctg
     3481 gggcaaacca gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt gaagggcaat
     3541 cagctgttgc ccgtctcact ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc
     3601 gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg
     3661 gaaagcgggc agtgagcgca acgcaattaa tgtaagttag ctcactcatt aggcaccggg
     3721 atctcgaccg atgcccttga gagccttcaa cccagtcagc tccttccggt gggcgcgggg
     3781 catgactatc gtcgccgcac ttatgactgt cttctttatc atgcaactcg taggacaggt
     3841 gccggcagcg ctctgggtca ttttcggcga ggaccgcttt cgctggagcg cgacgatgat
     3901 cggcctgtcg cttgcggtat tcggaatctt gcacgccctc gctcaagcct tcgtcactgg
     3961 tcccgccacc aaacgtttcg gcgagaagca ggccattatc gccggcatgg cggccccacg
     4021 ggtgcgcatg atcgtgctcc tgtcgttgag gacccggcta ggctggcggg gttgccttac
     4081 tggttagcag aatgaatcac cgatacgcga gcgaacgtga agcgactgct gctgcaaaac
     4141 gtctgcgacc tgagcaacaa catgaatggt cttcggtttc cgtgtttcgt aaagtctgga
     4201 aacgcggaag tcagcgccct gcaccattat gttccggatc tgcatcgcag gatgctgctg
     4261 gctaccctgt ggaacaccta catctgtatt aacgaagcgc tggcattgac cctgagtgat
     4321 ttttctctgg tcccgccgca tccataccgc cagttgttta ccctcacaac gttccagtaa
     4381 ccgggcatgt tcatcatcag taacccgtat cgtgagcatc ctctctcgtt tcatcggtat
     4441 cattaccccc atgaacagaa atccccctta cacggaggca tcagtgacca aacaggaaaa
     4501 aaccgccctt aacatggccc gctttatcag aagccagaca ttaacgcttc tggagaaact
     4561 caacgagctg gacgcggatg aacaggcaga catctgtgaa tcgcttcacg accacgctga
     4621 tgagctttac cgcagctgcc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat
     4681 gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg
     4741 tcagggcgcg tcagcgggtg ttggcgggtg tcggggcgca gccatgaccc agtcacgtag
     4801 cgatagcgga gtgtatactg gcttaactat gcggcatcag agcagattgt actgagagtg
     4861 caccattgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgct
     4921 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     4981 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     5041 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     5101 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     5161 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     5221 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     5281 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     5341 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     5401 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     5461 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     5521 ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta
     5581 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     5641 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     5701 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     5761 tcatgaacaa taaaactgtc tgcttacata aacagtaata caaggggtgt tatgagccat
     5821 attcaacggg aaacgtcttg ctctaggccg cgattaaatt ccaacatgga tgctgattta
     5881 tatgggtata aatgggctcg cgataatgtc gggcaatcag gtgcgacaat ctatcgattg
     5941 tatgggaagc ccgatgcgcc agagttgttt ctgaaacatg gcaaaggtag cgttgccaat
     6001 gatgttacag atgagatggt cagactaaac tggctgacgg aatttatgcc tcttccgacc
     6061 atcaagcatt ttatccgtac tcctgatgat gcatggttac tcaccactgc gatccccggg
     6121 aaaacagcat tccaggtatt agaagaatat cctgattcag gtgaaaatat tgttgatgcg
     6181 ctggcagtgt tcctgcgccg gttgcattcg attcctgttt gtaattgtcc ttttaacagc
     6241 gatcgcgtat ttcgtctcgc tcaggcgcaa tcacgaatga ataacggttt ggttgatgcg
     6301 agtgattttg atgacgagcg taatggctgg cctgttgaac aagtctggaa agaaatgcat
     6361 aaacttttgc cattctcacc ggattcagtc gtcactcatg gtgatttctc acttgataac
     6421 cttatttttg acgaggggaa attaataggt tgtattgatg ttggacgagt cggaatcgca
     6481 gaccgatacc aggatcttgc catcctatgg aactgcctcg gtgagttttc tccttcatta
     6541 cagaaacggc tttttcaaaa atatggtatt gataatcctg atatgaataa attgcagttt
     6601 catttgatgc tcgatgagtt tttctaagaa ttaattcatg agcggataca tatttgaatg
     6661 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctaa
     6721 attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt
     6781 tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata
     6841 gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac
     6901 gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa
     6961 tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc
     7021 cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg
     7081 aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca
     7141 cccgccgcgc ttaatgcgcc gctacagggc gcgtcccatt cgccaatc
//
