LOCUS       sequence-457046-        5940 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For expression of T. elongatus KaiB protein mutant.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5940)
  TITLE     LiWang lab plasmids
REFERENCE   2  (bases 1 to 5940)
  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-TeKaiB-1-94-Y8A-Y94A-FLAG
FEATURES             Location/Qualifiers
     source          1..5940
                     /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(182..205)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(488..511)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(512..805)
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             complement(818..835)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(845..862)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             881..903
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    918..942
                     /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(942..961)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(943..961)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1045..1064)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1270..1347
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1348..2430
                     /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(1367..1386)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3239..3430
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3424..3446)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3532..3671
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3587..3606
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3686..3703)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3857..4445)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3937..3956)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4556..4575)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4567..5382
                     /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             4567..5382
                     /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(4640..4659)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5474..5929)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5611..5632)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5823..5842
                     /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 atttgtcatc atcgtcttta tagtcagcca agagatctaa gccaatgagc accttctcac
      241 gattcgacaa gtccccaata atccggcgca cagggggcgg taggactttg gcaagggtag
      301 gcgtggccaa aattttatct tcctcagcca gttgcggatt tttgaggaca tcgattactt
      361 tgagtgcata gactccctta aattcttttt caagaatgtt attgagagtt tttagggcac
      421 gcaccgagtt gggtgtgtta ccggcaacgt atagcttgag aacagcggtt ttccgcaagg
      481 gggccatctt atcgtcgtca tccttgtaat ctccaccaat ctgttctctg tgagcctcaa
      541 taatatcgtt atcctccatg tccaaatctt caggggtctg atcagcttgg attctaatac
      601 cgtcgtacaa gaatcttaag gagtccattt ccttaccctg tcttttagcg aacgcttcca
      661 tcagccttct taaaggagtg gtctttttga tcttgaagaa gatctctgaa gatccatcgg
      721 acacctttaa attgatgtga gtctcaggct tgacttctgg cttgacctct ggcttagctt
      781 cttgattgac ttctgagtcc gacatgctag ccatatggct gccgcgcggc accaggccgc
      841 tgctgtgatg atgatgatga tggctgctgc ccatggtata tctccttctt aaagttaaac
      901 aaaattattt ctagagggga attgttatcc gctcacaatt cccctatagt gagtcgtatt
      961 aatttcgcgg gatcgagatc tcgatcctct acgccggacg catcgtggcc ggcatcaccg
     1021 gcgccacagg tgcggttgct ggcgcctata tcgccgacat caccgatggg gaagatcggg
     1081 ctcgccactt cgggctcatg agcgcttgtt tcggcgtggg tatggtggca ggccccgtgg
     1141 ccgggggact gttgggcgcc atctccttgc atgcaccatt ccttgcggcg gcggtgctca
     1201 acggcctcaa cctactactg ggctgcttcc taatgcagga gtcgcataag ggagagcgtc
     1261 gagatcccgg acaccatcga atggcgcaaa acctttcgcg gtatggcatg atagcgcccg
     1321 gaagagagtc aattcagggt ggtgaatgtg aaaccagtaa cgttatacga tgtcgcagag
     1381 tatgccggtg tctcttatca gaccgtttcc cgcgtggtga accaggccag ccacgtttct
     1441 gcgaaaacgc gggaaaaagt ggaagcggcg atggcggagc tgaattacat tcccaaccgc
     1501 gtggcacaac aactggcggg caaacagtcg ttgctgattg gcgttgccac ctccagtctg
     1561 gccctgcacg cgccgtcgca aattgtcgcg gcgattaaat ctcgcgccga tcaactgggt
     1621 gccagcgtgg tggtgtcgat ggtagaacga agcggcgtcg aagcctgtaa agcggcggtg
     1681 cacaatcttc tcgcgcaacg cgtcagtggg ctgatcatta actatccgct ggatgaccag
     1741 gatgccattg ctgtggaagc tgcctgcact aatgttccgg cgttatttct tgatgtctct
     1801 gaccagacac ccatcaacag tattattttc tcccatgaag acggtacgcg actgggcgtg
     1861 gagcatctgg tcgcattggg tcaccagcaa atcgcgctgt tagcgggccc attaagttct
     1921 gtctcggcgc gtctgcgtct ggctggctgg cataaatatc tcactcgcaa tcaaattcag
     1981 ccgatagcgg aacgggaagg cgactggagt gccatgtccg gttttcaaca aaccatgcaa
     2041 atgctgaatg agggcatcgt tcccactgcg atgctggttg ccaacgatca gatggcgctg
     2101 ggcgcaatgc gcgccattac cgagtccggg ctgcgcgttg gtgcggatat ctcggtagtg
     2161 ggatacgacg ataccgaaga cagctcatgt tatatcccgc cgttaaccac catcaaacag
     2221 gattttcgcc tgctggggca aaccagcgtg gaccgcttgc tgcaactctc tcagggccag
     2281 gcggtgaagg gcaatcagct gttgcccgtc tcactggtga aaagaaaaac caccctggcg
     2341 cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga
     2401 caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtaa gttagctcac
     2461 tcattaggca ccgggatctc gaccgatgcc cttgagagcc ttcaacccag tcagctcctt
     2521 ccggtgggcg cggggcatga ctatcgtcgc cgcacttatg actgtcttct ttatcatgca
     2581 actcgtagga caggtgccgg cagcgctctg ggtcattttc ggcgaggacc gctttcgctg
     2641 gagcgcgacg atgatcggcc tgtcgcttgc ggtattcgga atcttgcacg ccctcgctca
     2701 agccttcgtc actggtcccg ccaccaaacg tttcggcgag aagcaggcca ttatcgccgg
     2761 catggcggcc ccacgggtgc gcatgatcgt gctcctgtcg ttgaggaccc ggctaggctg
     2821 gcggggttgc cttactggtt agcagaatga atcaccgata cgcgagcgaa cgtgaagcga
     2881 ctgctgctgc aaaacgtctg cgacctgagc aacaacatga atggtcttcg gtttccgtgt
     2941 ttcgtaaagt ctggaaacgc ggaagtcagc gccctgcacc attatgttcc ggatctgcat
     3001 cgcaggatgc tgctggctac cctgtggaac acctacatct gtattaacga agcgctggca
     3061 ttgaccctga gtgatttttc tctggtcccg ccgcatccat accgccagtt gtttaccctc
     3121 acaacgttcc agtaaccggg catgttcatc atcagtaacc cgtatcgtga gcatcctctc
     3181 tcgtttcatc ggtatcatta cccccatgaa cagaaatccc ccttacacgg aggcatcagt
     3241 gaccaaacag gaaaaaaccg cccttaacat ggcccgcttt atcagaagcc agacattaac
     3301 gcttctggag aaactcaacg agctggacgc ggatgaacag gcagacatct gtgaatcgct
     3361 tcacgaccac gctgatgagc tttaccgcag ctgcctcgcg cgtttcggtg atgacggtga
     3421 aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg
     3481 gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat
     3541 gacccagtca cgtagcgata gcggagtgta tactggctta actatgcggc atcagagcag
     3601 attgtactga gagtgcacca ttgcggtgtg aaataccgca cagatgcgta aggagaaaat
     3661 accgcatcag gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     3721 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     3781 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     3841 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     3901 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     3961 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     4021 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     4081 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     4141 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     4201 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     4261 tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc
     4321 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     4381 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     4441 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     4501 gttaagggat tttggtcatg aacaataaaa ctgtctgctt acataaacag taatacaagg
     4561 ggtgttatga gccatattca acgggaaacg tcttgctcta ggccgcgatt aaattccaac
     4621 atggatgctg atttatatgg gtataaatgg gctcgcgata atgtcgggca atcaggtgcg
     4681 acaatctatc gattgtatgg gaagcccgat gcgccagagt tgtttctgaa acatggcaaa
     4741 ggtagcgttg ccaatgatgt tacagatgag atggtcagac taaactggct gacggaattt
     4801 atgcctcttc cgaccatcaa gcattttatc cgtactcctg atgatgcatg gttactcacc
     4861 actgcgatcc ccgggaaaac agcattccag gtattagaag aatatcctga ttcaggtgaa
     4921 aatattgttg atgcgctggc agtgttcctg cgccggttgc attcgattcc tgtttgtaat
     4981 tgtcctttta acagcgatcg cgtatttcgt ctcgctcagg cgcaatcacg aatgaataac
     5041 ggtttggttg atgcgagtga ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc
     5101 tggaaagaaa tgcataaact tttgccattc tcaccggatt cagtcgtcac tcatggtgat
     5161 ttctcacttg ataaccttat ttttgacgag gggaaattaa taggttgtat tgatgttgga
     5221 cgagtcggaa tcgcagaccg ataccaggat cttgccatcc tatggaactg cctcggtgag
     5281 ttttctcctt cattacagaa acggcttttt caaaaatatg gtattgataa tcctgatatg
     5341 aataaattgc agtttcattt gatgctcgat gagtttttct aagaattaat tcatgagcgg
     5401 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     5461 aaaagtgcca cctaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt
     5521 taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa
     5581 gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag
     5641 aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt
     5701 gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac
     5761 cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag
     5821 gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg
     5881 cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc ccattcgcca
//
