LOCUS       sequence-271357-        6206 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of recombinant Arabidopsis thaliana GUN1 (amino
            acids 709 to 918).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6206)
  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 6206)
  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-SMR1
FEATURES             Location/Qualifiers
     source          1..6206
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     132..151
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     protein_bind    258..282
                     /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             297..319
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             327..653
                     /codon_start=1
                     /gene="trxA"
                     /product="E. coli thioredoxin"
                     /label=TrxA
                     /translation="MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDE
                     IADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFL
                     DANLA"
     CDS             735..752
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             762..785
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             1469..1486
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1487..1531
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1612..1630)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1626..1673
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1710..2165
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1797..1816)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2007..2028
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2257..3072)
                     /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(2257..3072)
                     /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     2980..2999
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3064..3083
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3194..3782
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3683..3702
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3936..3953
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3968..4107
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4033..4052)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4193..4215
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4209..4400)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4972..6054)
                     /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
                     ALADSLMQLVRQVSRLESGQ"
     primer_bind     6016..6035
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6055..6132)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg
       61 gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga
      121 tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg
      181 atgccggcca cgatgcgtcc ggcgtagagg atcgagatcg atctcgatcc cgcgaaatta
      241 atacaactca ctatagggga attgtgagcg gataacaatt cccctctaga aataattttg
      301 tttaacttta agaaggagat atacatatga gcgataaaat tattcacctg actgacgaca
      361 gttttgacac ggatgtactc aaagcggacg gggcgatcct cgtcgatttc tgggcagagt
      421 ggtgcggtcc gtgcaaaatg atcgccccga ttctggatga aatcgctgac gaatatcagg
      481 gcaaactgac cgttgcaaaa ctgaacatcg atcaaaaccc tggcactgcg ccgaaatatg
      541 gcatccgtgg tatcccgact ctgctgctgt tcaaaaacgg tgaagtggcg gcaaccaaag
      601 tgggtgcact gtctaaaggt cagttgaaag agttcctcga cgctaacctg gccggttctg
      661 gttctggcca tactagtggt ggtggcggtt ctaataacaa tcctcctact cctactccat
      721 ctagtggttc tggtcatcac catcaccatc actccgcggc tcttgaagtc ctctttcagg
      781 gacccgggta ccaggatccg aattcatatg gtgttgttca tggacttctc atgggccaga
      841 gagagaatgt atggctccaa gctcagagct tatttgacaa agtaaatgaa atggatggtt
      901 cgactgcttc tgccttctac aatgccctaa ctgacatgct ttggcacttt ggccagaaac
      961 gaggtgcaga actggtggca cttgagggaa gatctaggca agtttgggaa aacgtgtggt
     1021 ctgattcttg cttggactta cacctcatgt cttctggagc tgcacgtgca atggtccatg
     1081 cgtggctact aaacatacgc tccattgtct atgaaggtca tgagttacct aaagtcttga
     1141 gtatattgac tggctggggg aaacacagca aagtggttgg agacggagca ctaagacgag
     1201 cggttgaagt tcttttgagg ggaatggatg caccattcca cctgtcaaaa tgcaacatgg
     1261 gacggttcac atcaagcggt tcggtggtag caacttggct gcgtgaatca gccacactga
     1321 aactcttaat cctccatgac cacataacaa ccgccacagc tactactaca acaatgaaat
     1381 caacagacca acaacaacgg aaacagacgt cgtttgcttt acagcctctt cttttgtagt
     1441 cgacaagctt gcggccgcag agctcgctct ggtgccacgc ggtagtaaag aaaccgctgc
     1501 tgctaaattc gaacgccagc acatggacag ctctacttct gctgctctcg aggcttaatt
     1561 aacctaggct gctaaacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc
     1621 aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag
     1681 gaggaactat atccggattg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc
     1741 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     1801 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     1861 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     1921 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     1981 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     2041 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     2101 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac
     2161 aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     2221 atacattcaa atatgtatcc gctcatgaat taattcttag aaaaactcat cgagcatcaa
     2281 atgaaactgc aatttattca tatcaggatt atcaatacca tatttttgaa aaagccgttt
     2341 ctgtaatgaa ggagaaaact caccgaggca gttccatagg atggcaagat cctggtatcg
     2401 gtctgcgatt ccgactcgtc caacatcaat acaacctatt aatttcccct cgtcaaaaat
     2461 aaggttatca agtgagaaat caccatgagt gacgactgaa tccggtgaga atggcaaaag
     2521 tttatgcatt tctttccaga cttgttcaac aggccagcca ttacgctcgt catcaaaatc
     2581 actcgcatca accaaaccgt tattcattcg tgattgcgcc tgagcgagac gaaatacgcg
     2641 atcactgtta aaaggacaat tacaaacagg aatcgaatgc aaccggcgca ggaacactgc
     2701 cagcgcatca acaatatttt cacctgaatc aggatattct tctaatacct ggaatgctgt
     2761 tttgccgggg atcgcagtgg tgagtaacca tgcatcatca ggagtacgga taaaatgctt
     2821 gatggtcgga agaggcataa attccgtcag ccagtttagt ctgaccatct catctgtaac
     2881 atcattggca acgctacctt tgccatgttt cagaaacaac tctggcgcat cgggcttccc
     2941 atacaatcga tagattgtcg cacctgattg cccgacatta tcgcgagccc atttataccc
     3001 atataaatca gcatccatgt tggaatttaa tcgcggccta gagcaagacg tttcccgttg
     3061 aatatggctc ataacacccc ttgtattact gtttatgtaa gcagacagtt ttattgttca
     3121 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     3181 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     3241 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     3301 aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt
     3361 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     3421 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     3481 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     3541 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     3601 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     3661 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     3721 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     3781 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     3841 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     3901 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     3961 aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcaatg
     4021 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata cactccgcta
     4081 tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc
     4141 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc
     4201 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc
     4261 tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct gttcatccgc gtccagctcg
     4321 ttgagtttct ccagaagcgt taatgtctgg cttctgataa agcgggccat gttaagggcg
     4381 gttttttcct gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt catgggggta
     4441 atgataccga tgaaacgaga gaggatgctc acgatacggg ttactgatga tgaacatgcc
     4501 cggttactgg aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga
     4561 aaaatcactc agggtcaatg ccagcgcttc gttaatacag atgtaggtgt tccacagggt
     4621 agccagcagc atcctgcgat gcagatccgg aacataatgg tgcagggcgc tgacttccgc
     4681 gtttccagac tttacgaaac acggaaaccg aagaccattc atgttgttgc tcaggtcgca
     4741 gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc attctgctaa
     4801 ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg acaggagcac gatcatgcta
     4861 gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt
     4921 cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc
     4981 gctttccagt cgggaaacct gtcgtaccag ctgcattaat gaatcggcca acgcgcgggg
     5041 agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa
     5101 cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt
     5161 ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct
     5221 gtcttcggta tcgtcgtatc ccactaccga gatgtccgca ccaacgcgca gcccggactc
     5281 ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg
     5341 aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc
     5401 gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc
     5461 cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg
     5521 acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat
     5581 actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc
     5641 agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac
     5701 gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac
     5761 catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat
     5821 ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt
     5881 gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc
     5941 cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt
     6001 ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac
     6061 caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca
     6121 ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc
     6181 ccagtagtag gttgaggccg ttgagc
//
