LOCUS       sequence-271365-        8328 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of recombinant Arabidopsis thaliana GUN1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8328)
  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 8328)
  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_full
FEATURES             Location/Qualifiers
     source          1..8328
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             136..153
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             163..186
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2992..3009
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             3010..3054
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(3135..3153)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3149..3196
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3233..3688
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3320..3339)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3530..3551
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3780..4595)
                     /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(3780..4595)
                     /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     4503..4522
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4587..4606
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4717..5305
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5206..5225
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5459..5476
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5491..5630
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5556..5575)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5716..5738
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5732..5923)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6495..7577)
                     /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     7539..7558
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7578..7655)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7861..7880
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     7968..7987
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        7968..7986
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    7987..8011
                     /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             8026..8048
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             join(8056..8328,1..54)
                     /codon_start=1
                     /gene="trxA"
                     /product="E. coli thioredoxin"
                     /label=TrxA
                     /translation="MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDE
                     IADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFL
                     DANLA"
ORIGIN
        1 gtgggtgcac tgtctaaagg tcagttgaaa gagttcctcg acgctaacct ggccggttct
       61 ggttctggcc atactagtgg tggtggcggt tctaataaca atcctcctac tcctactcca
      121 tctagtggtt ctggtcatca ccatcaccat cactccgcgg ctcttgaagt cctctttcag
      181 ggacccgggt accaggatcc gaattcggcg tcaacgccgc ctcactgggt caccaccacc
      241 aacaaccacc gtccatggct tcctcagcgt cctcgtcccg gtcgctccgt tacctcagct
      301 cctccctctt cttcagcctc tgtttcttca gctcatcttt cacatgacta ctccaaattt
      361 ctctcctttg caaaccccca aatcagattt ctccggtcgt caatctaccc gattcgtctc
      421 ccccgccacc aataaccacc gtcagaccag gcaaaaccca aactacaacc accgtccgta
      481 tggtgcctct tcttctcctc gtggctcagc tcctccgcct tcatctgtag ctactgtagc
      541 tccagctcaa ctatcacaac ctcctaactt ttcgcctctc caaaccccaa aatccgatct
      601 ttcatcggat ttttcaggta gacgatcgac tagattcgtc tccaagatgc atttcgggcg
      661 gcagaagacg acaatggcta cgagacatag ctctgctgct gaagatgcac ttcaaaacgc
      721 tattgatttc tctggagacg atgagatgtt tcatagtcta atgttgagct ttgagtcaaa
      781 gctgtgtggc tctgacgact gtacctatat aatccgtgag ttagggaatc gtaatgagtg
      841 cgataaagct gttggctttt atgagtttgc ggttaaacga gagaggagaa agaacgagca
      901 ggggaagtta gctagtgcta tgataagtac tcttggtaga tatggtaaag taactattgc
      961 taagaggatt ttcgaaactg cttttgctgg tggatatgga aacacggttt acgccttctc
     1021 ggctttgatc agcgcttatg ggagaagtgg gctacatgag gaagccatta gtgtcttcaa
     1081 ctccatgaaa gagtatggct tgaggccgaa tttggttacc tacaatgctg tgattgacgc
     1141 ttgcggtaaa ggcgggatgg agtttaagca agtagccaag tttttcgatg agatgcagag
     1201 gaacggtgtg cagccagata gaataacttt caactcgttg cttgctgttt gcagtagagg
     1261 agggttgtgg gaagcagcaa ggaatctgtt tgatgagatg acgaatagaa ggattgagca
     1321 agatgttttt tcttataata cgcttttgga tgcaatttgc aaaggtgggc agatggacct
     1381 tgctttcgaa atccttgctc agatgccagt taagagaatc atgcctaatg ttgtaagtta
     1441 tagcaccgtt attgatgggt ttgcaaaagc cggaagattt gatgaagctc ttaacttgtt
     1501 tggtgaaatg agatatcttg gtattgcatt agatagagtt tcttacaata cattgctttc
     1561 aatctacact aaagttggta gatctgaaga agctttggat attctgagag aaatggcatc
     1621 tgttgggatt aaaaaggatg ttgtgactta taatgctctt cttggaggat atgggaaaca
     1681 agggaaatat gatgaagtga agaaagtttt cactgagatg aagcgagagc atgtgttgcc
     1741 taacttactg acttattcaa ctttgatcga tgggtactcg aaggggggtt tgtataaaga
     1801 ggcaatggag atatttaggg agtttaagag cgccggatta agagctgatg ttgttttgta
     1861 cagtgcattg attgatgcat tgtgcaagaa tgggttggtg ggttctgctg tttctttgat
     1921 cgatgagatg acaaaggaag gaattagtcc taatgttgta acttataatt ccataatcga
     1981 tgcctttggt cgttctgcaa ctatggacag atcagccgat tatagcaatg gtggatcttt
     2041 gcctttctct tcttctgctc tgagtgcatt gactgaaaca gagggtaatc gggtaataca
     2101 gttatttggg cagttaacaa ctgagagtaa caaccgaacg acgaaagatt gtgaggaggg
     2161 tatgcaggag ctctcctgta tattggaagt tttccgcaaa atgcatcagc tggaaattaa
     2221 accaaatgtt gtaacctttt cagcgattct gaatgcttgc agtcggtgta actcatttga
     2281 agatgcatca atgctactag aggagctcag gctgtttgat aataaggtat atggtgttgt
     2341 tcatggactt ctcatgggcc agagagagaa tgtatggctc caagctcaga gcttatttga
     2401 caaagtaaat gaaatggatg gttcgactgc ttctgccttc tacaatgccc taactgacat
     2461 gctttggcac tttggccaga aacgaggtgc agaactggtg gcacttgagg gaagatctag
     2521 gcaagtttgg gaaaacgtgt ggtctgattc ttgcttggac ttacacctca tgtcttctgg
     2581 agctgcacgt gcaatggtcc atgcgtggct actaaacata cgctccattg tctatgaagg
     2641 tcatgagtta cctaaagtct tgagtatatt gactggctgg gggaaacaca gcaaagtggt
     2701 tggagacgga gcactaagac gagcggttga agttcttttg aggggaatgg atgcaccatt
     2761 ccacctgtca aaatgcaaca tgggacggtt cacatcaagc ggttcggtgg tagcaacttg
     2821 gctgcgtgaa tcagccacac tgaaactctt aatcctccat gaccacataa caaccgccac
     2881 agctactact acaacaatga aatcaacaga ccaacaacaa cggaaacaga cgtcgtttgc
     2941 tttacagcct cttcttttgt agtcgacaag cttgcggccg cagagctcgc tctggtgcca
     3001 cgcggtagta aagaaaccgc tgctgctaaa ttcgaacgcc agcacatgga cagctctact
     3061 tctgctgctc tcgaggctta attaacctag gctgctaaac aaagcccgaa aggaagctga
     3121 gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt
     3181 cttgaggggt tttttgctga aaggaggaac tatatccgga ttggcgaatg ggacgcgccc
     3241 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     3301 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     3361 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     3421 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc
     3481 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     3541 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt
     3601 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     3661 tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa
     3721 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg aattaattct
     3781 tagaaaaact catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata
     3841 ccatattttt gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat
     3901 aggatggcaa gatcctggta tcggtctgcg attccgactc gtccaacatc aatacaacct
     3961 attaatttcc cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg agtgacgact
     4021 gaatccggtg agaatggcaa aagtttatgc atttctttcc agacttgttc aacaggccag
     4081 ccattacgct cgtcatcaaa atcactcgca tcaaccaaac cgttattcat tcgtgattgc
     4141 gcctgagcga gacgaaatac gcgatcactg ttaaaaggac aattacaaac aggaatcgaa
     4201 tgcaaccggc gcaggaacac tgccagcgca tcaacaatat tttcacctga atcaggatat
     4261 tcttctaata cctggaatgc tgttttgccg gggatcgcag tggtgagtaa ccatgcatca
     4321 tcaggagtac ggataaaatg cttgatggtc ggaagaggca taaattccgt cagccagttt
     4381 agtctgacca tctcatctgt aacatcattg gcaacgctac ctttgccatg tttcagaaac
     4441 aactctggcg catcgggctt cccatacaat cgatagattg tcgcacctga ttgcccgaca
     4501 ttatcgcgag cccatttata cccatataaa tcagcatcca tgttggaatt taatcgcggc
     4561 ctagagcaag acgtttcccg ttgaatatgg ctcataacac cccttgtatt actgtttatg
     4621 taagcagaca gttttattgt tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     4681 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     4741 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     4801 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     4861 tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     4921 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     4981 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     5041 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     5101 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     5161 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     5221 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     5281 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     5341 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     5401 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
     5461 cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct
     5521 gtgcggtatt tcacaccgca atggtgcact ctcagtacaa tctgctctga tgccgcatag
     5581 ttaagccagt atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc
     5641 cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac
     5701 aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac
     5761 gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg aagcgattca cagatgtctg
     5821 cctgttcatc cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga
     5881 taaagcgggc catgttaagg gcggtttttt cctgtttggt cactgatgcc tccgtgtaag
     5941 ggggatttct gttcatgggg gtaatgatac cgatgaaacg agagaggatg ctcacgatac
     6001 gggttactga tgatgaacat gcccggttac tggaacgttg tgagggtaaa caactggcgg
     6061 tatggatgcg gcgggaccag agaaaaatca ctcagggtca atgccagcgc ttcgttaata
     6121 cagatgtagg tgttccacag ggtagccagc agcatcctgc gatgcagatc cggaacataa
     6181 tggtgcaggg cgctgacttc cgcgtttcca gactttacga aacacggaaa ccgaagacca
     6241 ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc
     6301 gtatcggtga ttcattctgc taaccagtaa ggcaaccccg ccagcctagc cgggtcctca
     6361 acgacaggag cacgatcatg ctagtcatgc cccgcgccca ccggaaggag ctgactgggt
     6421 tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat
     6481 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     6541 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt
     6601 cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt
     6661 tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt
     6721 aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatgtcc
     6781 gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg
     6841 ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga
     6901 aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga
     6961 gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc
     7021 gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta
     7081 ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat
     7141 aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga
     7201 tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag
     7261 gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg
     7321 cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca
     7381 acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa
     7441 ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc
     7501 tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat
     7561 aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc
     7621 ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg
     7681 cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc
     7741 aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac
     7801 catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc
     7861 ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg gcgccggtga tgccggccac
     7921 gatgcgtccg gcgtagagga tcgagatcga tctcgatccc gcgaaattaa tacgactcac
     7981 tataggggaa ttgtgagcgg ataacaattc ccctctagaa ataattttgt ttaactttaa
     8041 gaaggagata tacatatgag cgataaaatt attcacctga ctgacgacag ttttgacacg
     8101 gatgtactca aagcggacgg ggcgatcctc gtcgatttct gggcagagtg gtgcggtccg
     8161 tgcaaaatga tcgccccgat tctggatgaa atcgctgacg aatatcaggg caaactgacc
     8221 gttgcaaaac tgaacatcga tcaaaaccct ggcactgcgc cgaaatatgg catccgtggt
     8281 atcccgactc tgctgctgtt caaaaacggt gaagtggcgg caaccaaa
//
