LOCUS       sequence-457227-        5982 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For expression of T. elongatus KaiB protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5982)
  TITLE     LiWang lab plasmids
REFERENCE   2  (bases 1 to 5982)
  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-FLAG
FEATURES             Location/Qualifiers
     source          1..5982
                     /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(179..202)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(527..550)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(551..844)
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             complement(857..874)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(884..901)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             920..942
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    957..981
                     /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(981..1000)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(982..1000)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1084..1103)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1309..1386
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1387..2469
                     /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(1406..1425)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3278..3469
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3463..3485)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3571..3710
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3626..3645
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3725..3742)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3896..4484)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3976..3995)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4595..4614)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4606..5421
                     /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             4606..5421
                     /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(4679..4698)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5513..5968)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5650..5671)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5862..5881
                     /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 tatcgtcgtc atccttgtaa tcttccaagc ctaagtcatc ctcggcttgg tcaccaatct
      241 cttcatacaa gagatctaag ccaatgagca ccttctcacg attcgacaag tccccaataa
      301 tccggcgcac agggggcggt aggactttgg caagggtagg cgtggccaaa attttatctt
      361 cctcagccag ttgcggattt ttgaggacat cgattacttt gagtgcatag actcccttaa
      421 attctttttc aagaatgtta ttgagagttt ttagggcacg caccgagttg ggtgtgttac
      481 cggcaacgta tagcttgaga acataggttt tccgcaaggg ggccatctta tcgtcgtcat
      541 ccttgtaatc tccaccaatc tgttctctgt gagcctcaat aatatcgtta tcctccatgt
      601 ccaaatcttc aggggtctga tcagcttgga ttctaatacc gtcgtacaag aatcttaagg
      661 agtccatttc cttaccctgt cttttagcga acgcttccat cagccttctt aaaggagtgg
      721 tctttttgat cttgaagaag atctctgaag atccatcgga cacctttaaa ttgatgtgag
      781 tctcaggctt gacttctggc ttgacctctg gcttagcttc ttgattgact tctgagtccg
      841 acatgctagc catatggctg ccgcgcggca ccaggccgct gctgtgatga tgatgatgat
      901 ggctgctgcc catggtatat ctccttctta aagttaaaca aaattatttc tagaggggaa
      961 ttgttatccg ctcacaattc ccctatagtg agtcgtatta atttcgcggg atcgagatct
     1021 cgatcctcta cgccggacgc atcgtggccg gcatcaccgg cgccacaggt gcggttgctg
     1081 gcgcctatat cgccgacatc accgatgggg aagatcgggc tcgccacttc gggctcatga
     1141 gcgcttgttt cggcgtgggt atggtggcag gccccgtggc cgggggactg ttgggcgcca
     1201 tctccttgca tgcaccattc cttgcggcgg cggtgctcaa cggcctcaac ctactactgg
     1261 gctgcttcct aatgcaggag tcgcataagg gagagcgtcg agatcccgga caccatcgaa
     1321 tggcgcaaaa cctttcgcgg tatggcatga tagcgcccgg aagagagtca attcagggtg
     1381 gtgaatgtga aaccagtaac gttatacgat gtcgcagagt atgccggtgt ctcttatcag
     1441 accgtttccc gcgtggtgaa ccaggccagc cacgtttctg cgaaaacgcg ggaaaaagtg
     1501 gaagcggcga tggcggagct gaattacatt cccaaccgcg tggcacaaca actggcgggc
     1561 aaacagtcgt tgctgattgg cgttgccacc tccagtctgg ccctgcacgc gccgtcgcaa
     1621 attgtcgcgg cgattaaatc tcgcgccgat caactgggtg ccagcgtggt ggtgtcgatg
     1681 gtagaacgaa gcggcgtcga agcctgtaaa gcggcggtgc acaatcttct cgcgcaacgc
     1741 gtcagtgggc tgatcattaa ctatccgctg gatgaccagg atgccattgc tgtggaagct
     1801 gcctgcacta atgttccggc gttatttctt gatgtctctg accagacacc catcaacagt
     1861 attattttct cccatgaaga cggtacgcga ctgggcgtgg agcatctggt cgcattgggt
     1921 caccagcaaa tcgcgctgtt agcgggccca ttaagttctg tctcggcgcg tctgcgtctg
     1981 gctggctggc ataaatatct cactcgcaat caaattcagc cgatagcgga acgggaaggc
     2041 gactggagtg ccatgtccgg ttttcaacaa accatgcaaa tgctgaatga gggcatcgtt
     2101 cccactgcga tgctggttgc caacgatcag atggcgctgg gcgcaatgcg cgccattacc
     2161 gagtccgggc tgcgcgttgg tgcggatatc tcggtagtgg gatacgacga taccgaagac
     2221 agctcatgtt atatcccgcc gttaaccacc atcaaacagg attttcgcct gctggggcaa
     2281 accagcgtgg accgcttgct gcaactctct cagggccagg cggtgaaggg caatcagctg
     2341 ttgcccgtct cactggtgaa aagaaaaacc accctggcgc ccaatacgca aaccgcctct
     2401 ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc
     2461 gggcagtgag cgcaacgcaa ttaatgtaag ttagctcact cattaggcac cgggatctcg
     2521 accgatgccc ttgagagcct tcaacccagt cagctccttc cggtgggcgc ggggcatgac
     2581 tatcgtcgcc gcacttatga ctgtcttctt tatcatgcaa ctcgtaggac aggtgccggc
     2641 agcgctctgg gtcattttcg gcgaggaccg ctttcgctgg agcgcgacga tgatcggcct
     2701 gtcgcttgcg gtattcggaa tcttgcacgc cctcgctcaa gccttcgtca ctggtcccgc
     2761 caccaaacgt ttcggcgaga agcaggccat tatcgccggc atggcggccc cacgggtgcg
     2821 catgatcgtg ctcctgtcgt tgaggacccg gctaggctgg cggggttgcc ttactggtta
     2881 gcagaatgaa tcaccgatac gcgagcgaac gtgaagcgac tgctgctgca aaacgtctgc
     2941 gacctgagca acaacatgaa tggtcttcgg tttccgtgtt tcgtaaagtc tggaaacgcg
     3001 gaagtcagcg ccctgcacca ttatgttccg gatctgcatc gcaggatgct gctggctacc
     3061 ctgtggaaca cctacatctg tattaacgaa gcgctggcat tgaccctgag tgatttttct
     3121 ctggtcccgc cgcatccata ccgccagttg tttaccctca caacgttcca gtaaccgggc
     3181 atgttcatca tcagtaaccc gtatcgtgag catcctctct cgtttcatcg gtatcattac
     3241 ccccatgaac agaaatcccc cttacacgga ggcatcagtg accaaacagg aaaaaaccgc
     3301 ccttaacatg gcccgcttta tcagaagcca gacattaacg cttctggaga aactcaacga
     3361 gctggacgcg gatgaacagg cagacatctg tgaatcgctt cacgaccacg ctgatgagct
     3421 ttaccgcagc tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct
     3481 cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg
     3541 cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac gtagcgatag
     3601 cggagtgtat actggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat
     3661 tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg cgctcttccg
     3721 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     3781 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     3841 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     3901 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     3961 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     4021 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     4081 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     4141 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     4201 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     4261 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     4321 acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     4381 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     4441 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     4501 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     4561 acaataaaac tgtctgctta cataaacagt aatacaaggg gtgttatgag ccatattcaa
     4621 cgggaaacgt cttgctctag gccgcgatta aattccaaca tggatgctga tttatatggg
     4681 tataaatggg ctcgcgataa tgtcgggcaa tcaggtgcga caatctatcg attgtatggg
     4741 aagcccgatg cgccagagtt gtttctgaaa catggcaaag gtagcgttgc caatgatgtt
     4801 acagatgaga tggtcagact aaactggctg acggaattta tgcctcttcc gaccatcaag
     4861 cattttatcc gtactcctga tgatgcatgg ttactcacca ctgcgatccc cgggaaaaca
     4921 gcattccagg tattagaaga atatcctgat tcaggtgaaa atattgttga tgcgctggca
     4981 gtgttcctgc gccggttgca ttcgattcct gtttgtaatt gtccttttaa cagcgatcgc
     5041 gtatttcgtc tcgctcaggc gcaatcacga atgaataacg gtttggttga tgcgagtgat
     5101 tttgatgacg agcgtaatgg ctggcctgtt gaacaagtct ggaaagaaat gcataaactt
     5161 ttgccattct caccggattc agtcgtcact catggtgatt tctcacttga taaccttatt
     5221 tttgacgagg ggaaattaat aggttgtatt gatgttggac gagtcggaat cgcagaccga
     5281 taccaggatc ttgccatcct atggaactgc ctcggtgagt tttctccttc attacagaaa
     5341 cggctttttc aaaaatatgg tattgataat cctgatatga ataaattgca gtttcatttg
     5401 atgctcgatg agtttttcta agaattaatt catgagcgga tacatatttg aatgtattta
     5461 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctaaattgta
     5521 agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac
     5581 caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg
     5641 agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa
     5701 gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt
     5761 tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt
     5821 agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga
     5881 gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc
     5941 gcgcttaatg cgccgctaca gggcgcgtcc cattcgccaa tc
//
