LOCUS       sequence-460016-        6507 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For expression of T. elongatus KaiA protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6507)
  TITLE     LiWang lab plasmids
REFERENCE   2  (bases 1 to 6507)
  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-TeKaiA-FLAG
FEATURES             Location/Qualifiers
     source          1..6507
                     /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(1052..1075)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(1076..1369)
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             complement(1382..1399)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(1409..1426)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1445..1467
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1482..1506
                     /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(1506..1525)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1507..1525)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1609..1628)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1834..1911
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1912..2994
                     /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(1931..1950)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3803..3994
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3988..4010)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4096..4235
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4151..4170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4250..4267)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4421..5009)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4501..4520)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5120..5139)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5131..5946
                     /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             5131..5946
                     /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(5204..5223)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(6038..6493)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6175..6196)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6387..6406
                     /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 agcttttact
      181 tatcgtcgtc atccttgtaa tccacctccc gcggaatgga gcgacgatac atttcgcaca
      241 gatgggcaat gacatcaatc agtgtgaggc gatagtcgag caaaatgtcc tcactgcggc
      301 cttcgagttt gagttgcttg gaaaaggtat ccatgagttc gacgtgaatc tctagcactt
      361 gggaaacaga aatatctgcg aagaatgctt tactaacaaa ctcgtcaata cgctcattaa
      421 ctttggcatc ggtgttaaag tattcaagga caatggtacg ataaattgag cgcagttcgt
      481 ccagcagctt gcgtttatcg gcaggggaca tccgtcggaa aaaaaatgct gtatctcgtt
      541 tgtaatagac ccccaggtag ccgagtcgct ccttcaattt ttgggaaaga cgatgctggg
      601 ggtcaacatt actgctctct ttgagggctg gcaggcgaaa caggacgtgg ggcggtactg
      661 cacatcctgg acagagcttg acaaatcccg tgatggcttc ctcaatttga cggttgagct
      721 gatccagttg atcggttgtc aaaatcactt cagcaatgtg atagtgggga ccggcgggtg
      781 ggggggctgg cgcggctgga aaaatgagga tggcgggcaa gagggtggcg ctgtggtgaa
      841 gataggtaat gatttgcgga gtcccctctc cccattctaa aattagacag gcgatgctat
      901 tgcgccgtgc ctctaaatag ttacaaaact ccctctcgct tgaaaagtaa accagctcat
      961 caatatcaga ggtgtgcaaa cgaactagtt cttggccaat cgcgggcgag taaactaagc
     1021 cacaaattgt gagtgcggtt gactgcgcca ccttatcgtc gtcatccttg taatctccac
     1081 caatctgttc tctgtgagcc tcaataatat cgttatcctc catgtccaaa tcttcagggg
     1141 tctgatcagc ttggattcta ataccgtcgt acaagaatct taaggagtcc atttccttac
     1201 cctgtctttt agcgaacgct tccatcagcc ttcttaaagg agtggtcttt ttgatcttga
     1261 agaagatctc tgaagatcca tcggacacct ttaaattgat gtgagtctca ggcttgactt
     1321 ctggcttgac ctctggctta gcttcttgat tgacttctga gtccgacatg ctagccatat
     1381 ggctgccgcg cggcaccagg ccgctgctgt gatgatgatg atgatggctg ctgcccatgg
     1441 tatatctcct tcttaaagtt aaacaaaatt atttctagag gggaattgtt atccgctcac
     1501 aattccccta tagtgagtcg tattaatttc gcgggatcga gatctcgatc ctctacgccg
     1561 gacgcatcgt ggccggcatc accggcgcca caggtgcggt tgctggcgcc tatatcgccg
     1621 acatcaccga tggggaagat cgggctcgcc acttcgggct catgagcgct tgtttcggcg
     1681 tgggtatggt ggcaggcccc gtggccgggg gactgttggg cgccatctcc ttgcatgcac
     1741 cattccttgc ggcggcggtg ctcaacggcc tcaacctact actgggctgc ttcctaatgc
     1801 aggagtcgca taagggagag cgtcgagatc ccggacacca tcgaatggcg caaaaccttt
     1861 cgcggtatgg catgatagcg cccggaagag agtcaattca gggtggtgaa tgtgaaacca
     1921 gtaacgttat acgatgtcgc agagtatgcc ggtgtctctt atcagaccgt ttcccgcgtg
     1981 gtgaaccagg ccagccacgt ttctgcgaaa acgcgggaaa aagtggaagc ggcgatggcg
     2041 gagctgaatt acattcccaa ccgcgtggca caacaactgg cgggcaaaca gtcgttgctg
     2101 attggcgttg ccacctccag tctggccctg cacgcgccgt cgcaaattgt cgcggcgatt
     2161 aaatctcgcg ccgatcaact gggtgccagc gtggtggtgt cgatggtaga acgaagcggc
     2221 gtcgaagcct gtaaagcggc ggtgcacaat cttctcgcgc aacgcgtcag tgggctgatc
     2281 attaactatc cgctggatga ccaggatgcc attgctgtgg aagctgcctg cactaatgtt
     2341 ccggcgttat ttcttgatgt ctctgaccag acacccatca acagtattat tttctcccat
     2401 gaagacggta cgcgactggg cgtggagcat ctggtcgcat tgggtcacca gcaaatcgcg
     2461 ctgttagcgg gcccattaag ttctgtctcg gcgcgtctgc gtctggctgg ctggcataaa
     2521 tatctcactc gcaatcaaat tcagccgata gcggaacggg aaggcgactg gagtgccatg
     2581 tccggttttc aacaaaccat gcaaatgctg aatgagggca tcgttcccac tgcgatgctg
     2641 gttgccaacg atcagatggc gctgggcgca atgcgcgcca ttaccgagtc cgggctgcgc
     2701 gttggtgcgg atatctcggt agtgggatac gacgataccg aagacagctc atgttatatc
     2761 ccgccgttaa ccaccatcaa acaggatttt cgcctgctgg ggcaaaccag cgtggaccgc
     2821 ttgctgcaac tctctcaggg ccaggcggtg aagggcaatc agctgttgcc cgtctcactg
     2881 gtgaaaagaa aaaccaccct ggcgcccaat acgcaaaccg cctctccccg cgcgttggcc
     2941 gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa
     3001 cgcaattaat gtaagttagc tcactcatta ggcaccggga tctcgaccga tgcccttgag
     3061 agccttcaac ccagtcagct ccttccggtg ggcgcggggc atgactatcg tcgccgcact
     3121 tatgactgtc ttctttatca tgcaactcgt aggacaggtg ccggcagcgc tctgggtcat
     3181 tttcggcgag gaccgctttc gctggagcgc gacgatgatc ggcctgtcgc ttgcggtatt
     3241 cggaatcttg cacgccctcg ctcaagcctt cgtcactggt cccgccacca aacgtttcgg
     3301 cgagaagcag gccattatcg ccggcatggc ggccccacgg gtgcgcatga tcgtgctcct
     3361 gtcgttgagg acccggctag gctggcgggg ttgccttact ggttagcaga atgaatcacc
     3421 gatacgcgag cgaacgtgaa gcgactgctg ctgcaaaacg tctgcgacct gagcaacaac
     3481 atgaatggtc ttcggtttcc gtgtttcgta aagtctggaa acgcggaagt cagcgccctg
     3541 caccattatg ttccggatct gcatcgcagg atgctgctgg ctaccctgtg gaacacctac
     3601 atctgtatta acgaagcgct ggcattgacc ctgagtgatt tttctctggt cccgccgcat
     3661 ccataccgcc agttgtttac cctcacaacg ttccagtaac cgggcatgtt catcatcagt
     3721 aacccgtatc gtgagcatcc tctctcgttt catcggtatc attaccccca tgaacagaaa
     3781 tcccccttac acggaggcat cagtgaccaa acaggaaaaa accgccctta acatggcccg
     3841 ctttatcaga agccagacat taacgcttct ggagaaactc aacgagctgg acgcggatga
     3901 acaggcagac atctgtgaat cgcttcacga ccacgctgat gagctttacc gcagctgcct
     3961 cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac
     4021 agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt
     4081 tggcgggtgt cggggcgcag ccatgaccca gtcacgtagc gatagcggag tgtatactgg
     4141 cttaactatg cggcatcaga gcagattgta ctgagagtgc accattgcgg tgtgaaatac
     4201 cgcacagatg cgtaaggaga aaataccgca tcaggcgctc ttccgcttcc tcgctcactg
     4261 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     4321 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     4381 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     4441 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     4501 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     4561 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     4621 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     4681 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     4741 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     4801 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     4861 ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     4921 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     4981 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     5041 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgaacaat aaaactgtct
     5101 gcttacataa acagtaatac aaggggtgtt atgagccata ttcaacggga aacgtcttgc
     5161 tctaggccgc gattaaattc caacatggat gctgatttat atgggtataa atgggctcgc
     5221 gataatgtcg ggcaatcagg tgcgacaatc tatcgattgt atgggaagcc cgatgcgcca
     5281 gagttgtttc tgaaacatgg caaaggtagc gttgccaatg atgttacaga tgagatggtc
     5341 agactaaact ggctgacgga atttatgcct cttccgacca tcaagcattt tatccgtact
     5401 cctgatgatg catggttact caccactgcg atccccggga aaacagcatt ccaggtatta
     5461 gaagaatatc ctgattcagg tgaaaatatt gttgatgcgc tggcagtgtt cctgcgccgg
     5521 ttgcattcga ttcctgtttg taattgtcct tttaacagcg atcgcgtatt tcgtctcgct
     5581 caggcgcaat cacgaatgaa taacggtttg gttgatgcga gtgattttga tgacgagcgt
     5641 aatggctggc ctgttgaaca agtctggaaa gaaatgcata aacttttgcc attctcaccg
     5701 gattcagtcg tcactcatgg tgatttctca cttgataacc ttatttttga cgaggggaaa
     5761 ttaataggtt gtattgatgt tggacgagtc ggaatcgcag accgatacca ggatcttgcc
     5821 atcctatgga actgcctcgg tgagttttct ccttcattac agaaacggct ttttcaaaaa
     5881 tatggtattg ataatcctga tatgaataaa ttgcagtttc atttgatgct cgatgagttt
     5941 ttctaagaat taattcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     6001 aggggttccg cgcacatttc cccgaaaagt gccacctaaa ttgtaagcgt taatattttg
     6061 ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc
     6121 ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt
     6181 tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc
     6241 tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg
     6301 tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga
     6361 aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg
     6421 ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg
     6481 ctacagggcg cgtcccattc gccaatc
//
