LOCUS       sequence-271354-        6741 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of recombinant Arabidopsis thaliana GUN1 (amino
            acids 232 to 619).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6741)
  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 6741)
  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-PPR2
FEATURES             Location/Qualifiers
     source          1..6741
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(1..78)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     284..303
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     391..410
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        391..409
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    410..434
                     /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             449..471
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             479..805
                     /codon_start=1
                     /gene="trxA"
                     /product="E. coli thioredoxin"
                     /label=TrxA
                     /translation="MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDE
                     IADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFL
                     DANLA"
     CDS             887..904
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             914..937
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2156..2173
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             2174..2218
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2299..2317)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2313..2360
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2397..2852
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2484..2503)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2694..2715
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2944..3759)
                     /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(2944..3759)
                     /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     3667..3686
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3751..3770
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3881..4469
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4370..4389
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4623..4640
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4655..4794
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4720..4739)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4880..4902
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4896..5087)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5659..6741)
                     /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     6703..6722
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt
       61 gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga
      121 agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca
      181 aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac
      241 aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat
      301 aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga
      361 ggatcgagat cgatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag
      421 cggataacaa ttcccctcta gaaataattt tgtttaactt taagaaggag atatacatat
      481 gagcgataaa attattcacc tgactgacga cagttttgac acggatgtac tcaaagcgga
      541 cggggcgatc ctcgtcgatt tctgggcaga gtggtgcggt ccgtgcaaaa tgatcgcccc
      601 gattctggat gaaatcgctg acgaatatca gggcaaactg accgttgcaa aactgaacat
      661 cgatcaaaac cctggcactg cgccgaaata tggcatccgt ggtatcccga ctctgctgct
      721 gttcaaaaac ggtgaagtgg cggcaaccaa agtgggtgca ctgtctaaag gtcagttgaa
      781 agagttcctc gacgctaacc tggccggttc tggttctggc catactagtg gtggtggcgg
      841 ttctaataac aatcctccta ctcctactcc atctagtggt tctggtcatc accatcacca
      901 tcactccgcg gctcttgaag tcctctttca gggacccggg taccaggatc cgaattcaca
      961 ggggaagtta gctagtgcta tgataagtac tcttggtaga tatggtaaag taactattgc
     1021 taagaggatt ttcgaaactg cttttgctgg tggatatgga aacacggttt acgccttctc
     1081 ggctttgatc agcgcttatg ggagaagtgg gctacatgag gaagccatta gtgtcttcaa
     1141 ctccatgaaa gagtatggct tgaggccgaa tttggttacc tacaatgctg tgattgacgc
     1201 ttgcggtaaa ggcgggatgg agtttaagca agtagccaag tttttcgatg agatgcagag
     1261 gaacggtgtg cagccagata gaataacttt caactcgttg cttgctgttt gcagtagagg
     1321 agggttgtgg gaagcagcaa ggaatctgtt tgatgagatg acgaatagaa ggattgagca
     1381 agatgttttt tcttataata cgcttttgga tgcaatttgc aaaggtgggc agatggacct
     1441 tgctttcgaa atccttgctc agatgccagt taagagaatc atgcctaatg ttgtaagtta
     1501 tagcaccgtt attgatgggt ttgcaaaagc cggaagattt gatgaagctc ttaacttgtt
     1561 tggtgaaatg agatatcttg gtattgcatt agatagagtt tcttacaata cattgctttc
     1621 aatctacact aaagttggta gatctgaaga agctttggat attctgagag aaatggcatc
     1681 tgttgggatt aaaaaggatg ttgtgactta taatgctctt cttggaggat atgggaaaca
     1741 agggaaatat gatgaagtga agaaagtttt cactgagatg aagcgagagc atgtgttgcc
     1801 taacttactg acttattcaa ctttgatcga tgggtactcg aaggggggtt tgtataaaga
     1861 ggcaatggag atatttaggg agtttaagag cgccggatta agagctgatg ttgttttgta
     1921 cagtgcattg attgatgcat tgtgcaagaa tgggttggtg ggttctgctg tttctttgat
     1981 cgatgagatg acaaaggaag gaattagtcc taatgttgta acttataatt ccataatcga
     2041 tgcctttggt cgttctgcaa ctatggacag atcagccgat tatagcaatg gtggatcttt
     2101 gcctttctct tcttctgctc tgtaggtcga caagcttgcg gccgcagagc tcgctctggt
     2161 gccacgcggt agtaaagaaa ccgctgctgc taaattcgaa cgccagcaca tggacagctc
     2221 tacttctgct gctctcgagg cttaattaac ctaggctgct aaacaaagcc cgaaaggaag
     2281 ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac
     2341 gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg aatgggacgc
     2401 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     2461 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     2521 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     2581 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     2641 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     2701 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     2761 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     2821 gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc
     2881 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgaattaa
     2941 ttcttagaaa aactcatcga gcatcaaatg aaactgcaat ttattcatat caggattatc
     3001 aataccatat ttttgaaaaa gccgtttctg taatgaagga gaaaactcac cgaggcagtt
     3061 ccataggatg gcaagatcct ggtatcggtc tgcgattccg actcgtccaa catcaataca
     3121 acctattaat ttcccctcgt caaaaataag gttatcaagt gagaaatcac catgagtgac
     3181 gactgaatcc ggtgagaatg gcaaaagttt atgcatttct ttccagactt gttcaacagg
     3241 ccagccatta cgctcgtcat caaaatcact cgcatcaacc aaaccgttat tcattcgtga
     3301 ttgcgcctga gcgagacgaa atacgcgatc actgttaaaa ggacaattac aaacaggaat
     3361 cgaatgcaac cggcgcagga acactgccag cgcatcaaca atattttcac ctgaatcagg
     3421 atattcttct aatacctgga atgctgtttt gccggggatc gcagtggtga gtaaccatgc
     3481 atcatcagga gtacggataa aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca
     3541 gtttagtctg accatctcat ctgtaacatc attggcaacg ctacctttgc catgtttcag
     3601 aaacaactct ggcgcatcgg gcttcccata caatcgatag attgtcgcac ctgattgccc
     3661 gacattatcg cgagcccatt tatacccata taaatcagca tccatgttgg aatttaatcg
     3721 cggcctagag caagacgttt cccgttgaat atggctcata acaccccttg tattactgtt
     3781 tatgtaagca gacagtttta ttgttcatga ccaaaatccc ttaacgtgag ttttcgttcc
     3841 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     3901 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     3961 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     4021 atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     4081 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     4141 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     4201 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     4261 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     4321 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     4381 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     4441 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     4501 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     4561 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     4621 gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc
     4681 atctgtgcgg tatttcacac cgcaatggtg cactctcagt acaatctgct ctgatgccgc
     4741 atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga
     4801 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     4861 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     4921 aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg
     4981 tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt
     5041 ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg
     5101 taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg
     5161 atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg
     5221 gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt
     5281 aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac
     5341 ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag
     5401 accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc
     5461 tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc
     5521 ctcaacgaca ggagcacgat catgctagtc atgccccgcg cccaccggaa ggagctgact
     5581 gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt
     5641 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     5701 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt
     5761 ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga
     5821 gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt
     5881 ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat
     5941 gtccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg
     6001 atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg
     6061 ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt
     6121 gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg
     6181 gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg
     6241 cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag
     6301 aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag
     6361 cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt
     6421 acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc
     6481 ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt
     6541 ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat
     6601 gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct
     6661 ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc
     6721 gtataacgtt actggtttca c
//
