LOCUS       sequence-457045-        6462 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For expression of S. elongatus KaiA protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6462)
  TITLE     LiWang lab plasmids
REFERENCE   2  (bases 1 to 6462)
  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-SeKaiA
FEATURES             Location/Qualifiers
     source          1..6462
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             58..1140
                     /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(77..96)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             1949..2140
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(2134..2156)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    2242..2381
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     2297..2316
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(2396..2413)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2567..3155)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2647..2666)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(3266..3285)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3277..4092
                     /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             3277..4092
                     /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(3350..3369)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(4184..4639)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4321..4342)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4533..4552
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(4676..4723)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     4719..4737
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(4790..4807)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(5684..5977)
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             complement(5990..6007)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(6017..6034)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             6053..6075
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    6090..6114
                     /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(6114..6133)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6115..6133)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6217..6236)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        join(6442..6462,1..57)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 acctttcgcg gtatggcatg atagcgcccg gaagagagtc aattcagggt ggtgaatgtg
       61 aaaccagtaa cgttatacga tgtcgcagag tatgccggtg tctcttatca gaccgtttcc
      121 cgcgtggtga accaggccag ccacgtttct gcgaaaacgc gggaaaaagt ggaagcggcg
      181 atggcggagc tgaattacat tcccaaccgc gtggcacaac aactggcggg caaacagtcg
      241 ttgctgattg gcgttgccac ctccagtctg gccctgcacg cgccgtcgca aattgtcgcg
      301 gcgattaaat ctcgcgccga tcaactgggt gccagcgtgg tggtgtcgat ggtagaacga
      361 agcggcgtcg aagcctgtaa agcggcggtg cacaatcttc tcgcgcaacg cgtcagtggg
      421 ctgatcatta actatccgct ggatgaccag gatgccattg ctgtggaagc tgcctgcact
      481 aatgttccgg cgttatttct tgatgtctct gaccagacac ccatcaacag tattattttc
      541 tcccatgaag acggtacgcg actgggcgtg gagcatctgg tcgcattggg tcaccagcaa
      601 atcgcgctgt tagcgggccc attaagttct gtctcggcgc gtctgcgtct ggctggctgg
      661 cataaatatc tcactcgcaa tcaaattcag ccgatagcgg aacgggaagg cgactggagt
      721 gccatgtccg gttttcaaca aaccatgcaa atgctgaatg agggcatcgt tcccactgcg
      781 atgctggttg ccaacgatca gatggcgctg ggcgcaatgc gcgccattac cgagtccggg
      841 ctgcgcgttg gtgcggatat ctcggtagtg ggatacgacg ataccgaaga cagctcatgt
      901 tatatcccgc cgttaaccac catcaaacag gattttcgcc tgctggggca aaccagcgtg
      961 gaccgcttgc tgcaactctc tcagggccag gcggtgaagg gcaatcagct gttgcccgtc
     1021 tcactggtga aaagaaaaac caccctggcg cccaatacgc aaaccgcctc tccccgcgcg
     1081 ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga
     1141 gcgcaacgca attaatgtaa gttagctcac tcattaggca ccgggatctc gaccgatgcc
     1201 cttgagagcc ttcaacccag tcagctcctt ccggtgggcg cggggcatga ctatcgtcgc
     1261 cgcacttatg actgtcttct ttatcatgca actcgtagga caggtgccgg cagcgctctg
     1321 ggtcattttc ggcgaggacc gctttcgctg gagcgcgacg atgatcggcc tgtcgcttgc
     1381 ggtattcgga atcttgcacg ccctcgctca agccttcgtc actggtcccg ccaccaaacg
     1441 tttcggcgag aagcaggcca ttatcgccgg catggcggcc ccacgggtgc gcatgatcgt
     1501 gctcctgtcg ttgaggaccc ggctaggctg gcggggttgc cttactggtt agcagaatga
     1561 atcaccgata cgcgagcgaa cgtgaagcga ctgctgctgc aaaacgtctg cgacctgagc
     1621 aacaacatga atggtcttcg gtttccgtgt ttcgtaaagt ctggaaacgc ggaagtcagc
     1681 gccctgcacc attatgttcc ggatctgcat cgcaggatgc tgctggctac cctgtggaac
     1741 acctacatct gtattaacga agcgctggca ttgaccctga gtgatttttc tctggtcccg
     1801 ccgcatccat accgccagtt gtttaccctc acaacgttcc agtaaccggg catgttcatc
     1861 atcagtaacc cgtatcgtga gcatcctctc tcgtttcatc ggtatcatta cccccatgaa
     1921 cagaaatccc ccttacacgg aggcatcagt gaccaaacag gaaaaaaccg cccttaacat
     1981 ggcccgcttt atcagaagcc agacattaac gcttctggag aaactcaacg agctggacgc
     2041 ggatgaacag gcagacatct gtgaatcgct tcacgaccac gctgatgagc tttaccgcag
     2101 ctgcctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac
     2161 ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc
     2221 gggtgttggc gggtgtcggg gcgcagccat gacccagtca cgtagcgata gcggagtgta
     2281 tactggctta actatgcggc atcagagcag attgtactga gagtgcacca ttgcggtgtg
     2341 aaataccgca cagatgcgta aggagaaaat accgcatcag gcgctcttcc gcttcctcgc
     2401 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     2461 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     2521 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     2581 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     2641 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     2701 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     2761 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     2821 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     2881 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     2941 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     3001 ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     3061 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     3121 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     3181 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg aacaataaaa
     3241 ctgtctgctt acataaacag taatacaagg ggtgttatga gccatattca acgggaaacg
     3301 tcttgctcta ggccgcgatt aaattccaac atggatgctg atttatatgg gtataaatgg
     3361 gctcgcgata atgtcgggca atcaggtgcg acaatctatc gattgtatgg gaagcccgat
     3421 gcgccagagt tgtttctgaa acatggcaaa ggtagcgttg ccaatgatgt tacagatgag
     3481 atggtcagac taaactggct gacggaattt atgcctcttc cgaccatcaa gcattttatc
     3541 cgtactcctg atgatgcatg gttactcacc actgcgatcc ccgggaaaac agcattccag
     3601 gtattagaag aatatcctga ttcaggtgaa aatattgttg atgcgctggc agtgttcctg
     3661 cgccggttgc attcgattcc tgtttgtaat tgtcctttta acagcgatcg cgtatttcgt
     3721 ctcgctcagg cgcaatcacg aatgaataac ggtttggttg atgcgagtga ttttgatgac
     3781 gagcgtaatg gctggcctgt tgaacaagtc tggaaagaaa tgcataaact tttgccattc
     3841 tcaccggatt cagtcgtcac tcatggtgat ttctcacttg ataaccttat ttttgacgag
     3901 gggaaattaa taggttgtat tgatgttgga cgagtcggaa tcgcagaccg ataccaggat
     3961 cttgccatcc tatggaactg cctcggtgag ttttctcctt cattacagaa acggcttttt
     4021 caaaaatatg gtattgataa tcctgatatg aataaattgc agtttcattt gatgctcgat
     4081 gagtttttct aagaattaat tcatgagcgg atacatattt gaatgtattt agaaaaataa
     4141 acaaataggg gttccgcgca catttccccg aaaagtgcca cctaaattgt aagcgttaat
     4201 attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc
     4261 gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt
     4321 ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa
     4381 accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg
     4441 tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga
     4501 cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct
     4561 agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat
     4621 gcgccgctac agggcgcgtc ccattcgcca atccggatat agttcctcct ttcagcaaaa
     4681 aacccctcaa gacccgttta gaggccccaa ggggttatgc tagttattgc tcagcggtgg
     4741 cagcagccaa ctcagcttcc tttcgggctt tgttagcagc cggatctcag tggtggtggt
     4801 ggtggtgctc gagtgcggcc gcaagctttc aggtttctcg tgggatagac cgtcgataca
     4861 tctcacaaag atgtgcaatt acatcaatta aagtcagccg ataatccagc aaaatgtcct
     4921 ctgaacgtcc ctctacgcgg agcttcttgg caaactcgtc catcagctcc atgtgaattt
     4981 ctaccacttt ggtgactgga acatcggcaa agaaagccat gttgacgaag ttgtcaatgc
     5041 tctggttgag gttgctattc ggcgaaaaat agctcaaaac gatttcacga tagctagtct
     5101 gcatcgcttg gtgcagcttt tggctttcgt aggcgggtag gttgcgcaga aagcgatcgg
     5161 gatcacgctt gtagtagacc ccgagatagc ctaggcgctc ttgtagtcgc tgagcgaggt
     5221 cccgctgctg gctcgatagc tcgggatcgt ggttggcccc catcagcatg atgtggtcgg
     5281 ccatggtctc gaccggggct aagcgcagaa attcagccag tgcagcatca acttggtagg
     5341 gcaattgctc gagctgatgg ataccgaggt gcagttcagc gctgtgatag agctgttctt
     5401 tggctggttc atcgggatcc tcactgtcgc gatcgcctac gacaatcgct ggtaccacca
     5461 ctccctcaaa gcagagctgc tgaacaactg ccctgaagct gggattggct gccactaaaa
     5521 tcaggcagtc gatttggtca cgatgggttt gggcatactc caagagcatt tcgccagact
     5581 cacagacttg gagttgatag cgatcggccg acagcgcccg ctggcaatcc tgcaaaattg
     5641 ccgtcgattc cacccaaatg cagattgcaa tttgcgagag cactccacca atctgttctc
     5701 tgtgagcctc aataatatcg ttatcctcca tgtccaaatc ttcaggggtc tgatcagctt
     5761 ggattctaat accgtcgtac aagaatctta aggagtccat ttccttaccc tgtcttttag
     5821 cgaacgcttc catcagcctt cttaaaggag tggtcttttt gatcttgaag aagatctctg
     5881 aagatccatc ggacaccttt aaattgatgt gagtctcagg cttgacttct ggcttgacct
     5941 ctggcttagc ttcttgattg acttctgagt ccgacatgct agccatatgg ctgccgcgcg
     6001 gcaccaggcc gctgctgtga tgatgatgat gatggctgct gcccatggta tatctccttc
     6061 ttaaagttaa acaaaattat ttctagaggg gaattgttat ccgctcacaa ttcccctata
     6121 gtgagtcgta ttaatttcgc gggatcgaga tctcgatcct ctacgccgga cgcatcgtgg
     6181 ccggcatcac cggcgccaca ggtgcggttg ctggcgccta tatcgccgac atcaccgatg
     6241 gggaagatcg ggctcgccac ttcgggctca tgagcgcttg tttcggcgtg ggtatggtgg
     6301 caggccccgt ggccggggga ctgttgggcg ccatctcctt gcatgcacca ttccttgcgg
     6361 cggcggtgct caacggcctc aacctactac tgggctgctt cctaatgcag gagtcgcata
     6421 agggagagcg tcgagatccc ggacaccatc gaatggcgca aa
//
