LOCUS       sequence-271363-        7008 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of recombinant Arabidopsis thaliana GUN1 (amino
            acids 232 to 708).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7008)
  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 7008)
  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-PPR1
FEATURES             Location/Qualifiers
     source          1..7008
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     96..115
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     203..222
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        203..221
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    222..246
                     /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             261..283
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             291..617
                     /codon_start=1
                     /gene="trxA"
                     /product="E. coli thioredoxin"
                     /label=TrxA
                     /translation="MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDE
                     IADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFL
                     DANLA"
     CDS             699..716
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             726..749
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2235..2252
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             2253..2297
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2378..2396)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2392..2439
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2476..2931
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2563..2582)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2773..2794
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3023..3838)
                     /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(3023..3838)
                     /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     3746..3765
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3830..3849
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3960..4548
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4449..4468
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4702..4719
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4734..4873
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4799..4818)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4959..4981
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4975..5166)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5738..6820)
                     /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
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSCADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     6782..6801
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6821..6898)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa acaagcgctc
       61 atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca
      121 gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggatcgag
      181 atcgatctcg atcccgcgaa attaatacga ctcactatag gggaattgtg agcggataac
      241 aattcccctc tagaaataat tttgtttaac tttaagaagg agatatacat atgagcgata
      301 aaattattca cctgactgac gacagttttg acacggatgt actcaaagcg gacggggcga
      361 tcctcgtcga tttctgggca gagtggtgcg gtccgtgcaa aatgatcgcc ccgattctgg
      421 atgaaatcgc tgacgaatat cagggcaaac tgaccgttgc aaaactgaac atcgatcaaa
      481 accctggcac tgcgccgaaa tatggcatcc gtggtatccc gactctgctg ctgttcaaaa
      541 acggtgaagt ggcggcaacc aaagtgggtg cactgtctaa aggtcagttg aaagagttcc
      601 tcgacgctaa cctggccggt tctggttctg gccatactag tggtggtggc ggttctaata
      661 acaatcctcc tactcctact ccatctagtg gttctggtca tcaccatcac catcactccg
      721 cggctcttga agtcctcttt cagggacccg ggtaccagga tccgaattca caggggaagt
      781 tagctagtgc tatgataagt actcttggta gatatggtaa agtaactatt gctaagagga
      841 ttttcgaaac tgcttttgct ggtggatatg gaaacacggt ttacgccttc tcggctttga
      901 tcagcgctta tgggagaagt gggctacatg aggaagccat tagtgtcttc aactccatga
      961 aagagtatgg cttgaggccg aatttggtta cctacaatgc tgtgattgac gcttgcggta
     1021 aaggcgggat ggagtttaag caagtagcca agtttttcga tgagatgcag aggaacggtg
     1081 tgcagccaga tagaataact ttcaactcgt tgcttgctgt ttgcagtaga ggagggttgt
     1141 gggaagcagc aaggaatctg tttgatgaga tgacgaatag aaggattgag caagatgttt
     1201 tttcttataa tacgcttttg gatgcaattt gcaaaggtgg gcagatggac cttgctttcg
     1261 aaatccttgc tcagatgcca gttaagagaa tcatgcctaa tgttgtaagt tatagcaccg
     1321 ttattgatgg gtttgcaaaa gccggaagat ttgatgaagc tcttaacttg tttggtgaaa
     1381 tgagatatct tggtattgca ttagatagag tttcttacaa tacattgctt tcaatctaca
     1441 ctaaagttgg tagatctgaa gaagctttgg atattctgag agaaatggca tctgttggga
     1501 ttaaaaagga tgttgtgact tataatgctc ttcttggagg atatgggaaa caagggaaat
     1561 atgatgaagt gaagaaagtt ttcactgaga tgaagcgaga gcatgtgttg cctaacttac
     1621 tgacttattc aactttgatc gatgggtact cgaagggggg tttgtataaa gaggcaatgg
     1681 agatatttag ggagtttaag agcgccggat taagagctga tgttgttttg tacagtgcat
     1741 tgattgatgc attgtgcaag aatgggttgg tgggttctgc tgtttctttg atcgatgaga
     1801 tgacaaagga aggaattagt cctaatgttg taacttataa ttccataatc gatgcctttg
     1861 gtcgttctgc aactatggac agatcagccg attatagcaa tggtggatct ttgcctttct
     1921 cttcttctgc tctgagtgca ttgactgaaa cagagggtaa tcgggtaata cagttatttg
     1981 ggcagttaac aactgagagt aacaaccgaa cgacgaaaga ttgtgaggag ggtatgcagg
     2041 agctctcctg tatattggaa gttttccgca aaatgcatca gctggaaatt aaaccaaatg
     2101 ttgtaacctt ttcagcgatt ctgaatgctt gcagtcggtg taactcattt gaagatgcat
     2161 caatgctact agaggagctc aggctgtttg ataataaggt ataggtcgac aagcttgcgg
     2221 ccgcagagct cgctctggtg ccacgcggta gtaaagaaac cgctgctgct aaattcgaac
     2281 gccagcacat ggacagctct acttctgctg ctctcgaggc ttaattaacc taggctgcta
     2341 aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa
     2401 ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc
     2461 ggattggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     2521 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     2581 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     2641 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     2701 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     2761 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     2821 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     2881 tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac
     2941 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     3001 gtatccgctc atgaattaat tcttagaaaa actcatcgag catcaaatga aactgcaatt
     3061 tattcatatc aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag
     3121 aaaactcacc gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga
     3181 ctcgtccaac atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg
     3241 agaaatcacc atgagtgacg actgaatccg gtgagaatgg caaaagttta tgcatttctt
     3301 tccagacttg ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca
     3361 aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa tacgcgatca ctgttaaaag
     3421 gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa
     3481 tattttcacc tgaatcagga tattcttcta atacctggaa tgctgttttg ccggggatcg
     3541 cagtggtgag taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag
     3601 gcataaattc cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc
     3661 tacctttgcc atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcgataga
     3721 ttgtcgcacc tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat
     3781 ccatgttgga atttaatcgc ggcctagagc aagacgtttc ccgttgaata tggctcataa
     3841 caccccttgt attactgttt atgtaagcag acagttttat tgttcatgac caaaatccct
     3901 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     3961 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     4021 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     4081 agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc
     4141 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     4201 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     4261 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     4321 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     4381 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     4441 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     4501 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     4561 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     4621 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     4681 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg
     4741 cggtattttc tccttacgca tctgtgcggt atttcacacc gcaatggtgc actctcagta
     4801 caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg
     4861 ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     4921 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
     4981 gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc
     5041 gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag
     5101 aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt
     5161 ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa
     5221 acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg
     5281 ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg
     5341 tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc
     5401 tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta
     5461 cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca
     5521 gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc
     5581 ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgctagtca tgccccgcgc
     5641 ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc
     5701 ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     5761 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     5821 tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct
     5881 tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc
     5941 gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt
     6001 cgtatcccac taccgagatg tccgcaccaa cgcgcagccc ggactcggta atggcgcgca
     6061 ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat
     6121 tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg
     6181 ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg
     6241 ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca
     6301 gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg
     6361 tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa
     6421 tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa
     6481 gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca
     6541 cgctggcacc cagttgatcg gcgcaagatt taatcgccgc gacaatttgc gacggcgcgt
     6601 gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt
     6661 gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg
     6721 ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac
     6781 cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac
     6841 tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg atggtgtccg
     6901 ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag tagtaggttg
     6961 aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatg
//
