LOCUS       sequence-271367-        7637 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of recombinant Arabidopsis thaliana GUN1 (amino
            acids 232 to 918).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7637)
  AUTHORS   Shimizu T, Kacprzak SM, Mochizuki N, Nagatani A, Watanabe S, Shimada
            T, Tanaka K, Hayashi Y, Arai M, Leister D, Okamoto H, Terry MJ, 
            Masuda T
  TITLE     The retrograde signaling protein GUN1 regulates tetrapyrrole 
            biosynthesis.
  JOURNAL   Proc Natl Acad Sci U S A. 2019 Dec 3;116(49):24900-24906. doi: 
            10.1073/pnas.1911251116. Epub 2019 Nov 15.
  PUBMED    31732672
REFERENCE   2  (bases 1 to 7637)
  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"; doi: 
            "10.1073/pnas.1911251116"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2019-12-3- 3"; volume: "116"; issue: "49"; pages: 
            "24900-24906"
COMMENT     Sequence Label: pET48 AtGUN1-PS
FEATURES             Location/Qualifiers
     source          1..7637
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     196..215
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     303..322
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        303..321
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    322..346
                     /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)."
     RBS             361..383
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             391..717
                     /codon_start=1
                     /gene="trxA"
                     /product="E. coli thioredoxin"
                     /label=TrxA
                     /translation="MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDE
                     IADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFL
                     DANLA"
     CDS             799..816
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             826..849
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2964..2981
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             2982..3026
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(3107..3125)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3121..3168
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3205..3660
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3292..3311)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3502..3523
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3752..4567)
                     /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             complement(3752..4567)
                     /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     4475..4494
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4559..4578
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4689..5277
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5178..5197
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5431..5448
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5463..5602
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5528..5547)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5688..5710
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5704..5895)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6467..7549)
                     /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     7511..7530
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7550..7627)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 acgctctccc ttatgcgact cctgcattag gaagcagccc agtagtaggt tgaggccgtt
       61 gagcaccgcc gccgcaagga atggtgcatg caaggagatg gcgcccaaca gtcccccggc
      121 cacggggcct gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcgagc
      181 ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc
      241 ggtgatgccg gccacgatgc gtccggcgta gaggatcgag atcgatctcg atcccgcgaa
      301 attaatacga ctcactatag gggaattgtg agcggataac aattcccctc tagaaataat
      361 tttgtttaac tttaagaagg agatatacat atgagcgata aaattattca cctgactgac
      421 gacagttttg acacggatgt actcaaagcg gacggggcga tcctcgtcga tttctgggca
      481 gagtggtgcg gtccgtgcaa aatgatcgcc ccgattctgg atgaaatcgc tgacgaatat
      541 cagggcaaac tgaccgttgc aaaactgaac atcgatcaaa accctggcac tgcgccgaaa
      601 tatggcatcc gtggtatccc gactctgctg ctgttcaaaa acggtgaagt ggcggcaacc
      661 aaagtgggtg cactgtctaa aggtcagttg aaagagttcc tcgacgctaa cctggccggt
      721 tctggttctg gccatactag tggtggtggc ggttctaata acaatcctcc tactcctact
      781 ccatctagtg gttctggtca tcaccatcac catcactccg cggctcttga agtcctcttt
      841 cagggacccg ggtaccagga tccgaattca caggggaagt tagctagtgc tatgataagt
      901 actcttggta gatatggtaa agtaactatt gctaagagga ttttcgaaac tgcttttgct
      961 ggtggatatg gaaacacggt ttacgccttc tcggctttga tcagcgctta tgggagaagt
     1021 gggctacatg aggaagccat tagtgtcttc aactccatga aagagtatgg cttgaggccg
     1081 aatttggtta cctacaatgc tgtgattgac gcttgcggta aaggcgggat ggagtttaag
     1141 caagtagcca agtttttcga tgagatgcag aggaacggtg tgcagccaga tagaataact
     1201 ttcaactcgt tgcttgctgt ttgcagtaga ggagggttgt gggaagcagc aaggaatctg
     1261 tttgatgaga tgacgaatag aaggattgag caagatgttt tttcttataa tacgcttttg
     1321 gatgcaattt gcaaaggtgg gcagatggac cttgctttcg aaatccttgc tcagatgcca
     1381 gttaagagaa tcatgcctaa tgttgtaagt tatagcaccg ttattgatgg gtttgcaaaa
     1441 gccggaagat ttgatgaagc tcttaacttg tttggtgaaa tgagatatct tggtattgca
     1501 ttagatagag tttcttacaa tacattgctt tcaatctaca ctaaagttgg tagatctgaa
     1561 gaagctttgg atattctgag agaaatggca tctgttggga ttaaaaagga tgttgtgact
     1621 tataatgctc ttcttggagg atatgggaaa caagggaaat atgatgaagt gaagaaagtt
     1681 ttcactgaga tgaagcgaga gcatgtgttg cctaacttac tgacttattc aactttgatc
     1741 gatgggtact cgaagggggg tttgtataaa gaggcaatgg agatatttag ggagtttaag
     1801 agcgccggat taagagctga tgttgttttg tacagtgcat tgattgatgc attgtgcaag
     1861 aatgggttgg tgggttctgc tgtttctttg atcgatgaga tgacaaagga aggaattagt
     1921 cctaatgttg taacttataa ttccataatc gatgcctttg gtcgttctgc aactatggac
     1981 agatcagccg attatagcaa tggtggatct ttgcctttct cttcttctgc tctgagtgca
     2041 ttgactgaaa cagagggtaa tcgggtaata cagttatttg ggcagttaac aactgagagt
     2101 aacaaccgaa cgacgaaaga ttgtgaggag ggtatgcagg agctctcctg tatattggaa
     2161 gttttccgca aaatgcatca gctggaaatt aaaccaaatg ttgtaacctt ttcagcgatt
     2221 ctgaatgctt gcagtcggtg taactcattt gaagatgcat caatgctact agaggagctc
     2281 aggctgtttg ataataaggt atatggtgtt gttcatggac ttctcatggg ccagagagag
     2341 aatgtatggc tccaagctca gagcttattt gacaaagtaa atgaaatgga tggttcgact
     2401 gcttctgcct tctacaatgc cctaactgac atgctttggc actttggcca gaaacgaggt
     2461 gcagaactgg tggcacttga gggaagatct aggcaagttt gggaaaacgt gtggtctgat
     2521 tcttgcttgg acttacacct catgtcttct ggagctgcac gtgcaatggt ccatgcgtgg
     2581 ctactaaaca tacgctccat tgtctatgaa ggtcatgagt tacctaaagt cttgagtata
     2641 ttgactggct gggggaaaca cagcaaagtg gttggagacg gagcactaag acgagcggtt
     2701 gaagttcttt tgaggggaat ggatgcacca ttccacctgt caaaatgcaa catgggacgg
     2761 ttcacatcaa gcggttcggt ggtagcaact tggctgcgtg aatcagccac actgaaactc
     2821 ttaatcctcc atgaccacat aacaaccgcc acagctacta ctacaacaat gaaatcaaca
     2881 gaccaacaac aacggaaaca gacgtcgttt gctttacagc ctcttctttt gtagtcgaca
     2941 agcttgcggc cgcagagctc gctctggtgc cacgcggtag taaagaaacc gctgctgcta
     3001 aattcgaacg ccagcacatg gacagctcta cttctgctgc tctcgaggct taattaacct
     3061 aggctgctaa acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa
     3121 ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga
     3181 actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     3241 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     3301 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     3361 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     3421 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     3481 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     3541 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     3601 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt
     3661 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     3721 ttcaaatatg tatccgctca tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa
     3781 actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta
     3841 atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg
     3901 cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt
     3961 tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagtttat
     4021 gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg
     4081 catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat acgcgatcac
     4141 tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg
     4201 catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat gctgttttgc
     4261 cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg
     4321 tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat
     4381 tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca
     4441 atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata
     4501 aatcagcatc catgttggaa tttaatcgcg gcctagagca agacgtttcc cgttgaatat
     4561 ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt gttcatgacc
     4621 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     4681 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     4741 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     4801 actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc
     4861 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     4921 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     4981 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     5041 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     5101 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     5161 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     5221 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     5281 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     5341 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     5401 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     5461 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg caatggtgca
     5521 ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct
     5581 acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg
     5641 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     5701 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc
     5761 agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag
     5821 tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt
     5881 ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat
     5941 accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt
     6001 actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat
     6061 cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca
     6121 gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc
     6181 cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt
     6241 tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt
     6301 aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca tgctagtcat
     6361 gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga
     6421 tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt
     6481 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     6541 cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct
     6601 gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc
     6661 ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt
     6721 cggtatcgtc gtatcccact accgagatgt ccgcaccaac gcgcagcccg gactcggtaa
     6781 tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga
     6841 tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt
     6901 cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac
     6961 gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca
     7021 atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt
     7081 tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt
     7141 ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt
     7201 gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg
     7261 acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg
     7321 acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg
     7381 ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt
     7441 tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat
     7501 aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc
     7561 tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga
     7621 tggtgtccgg gatctcg
//
