LOCUS       sequence-271171-        5990 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of recombinant Arabidopsis thaliana GUN1 (amino
            acids 781 to 918).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5990)
  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 5990)
  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-SMR2
FEATURES             Location/Qualifiers
     source          1..5990
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     158..177
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     265..284
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        265..283
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    284..308
                     /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             323..345
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             353..679
                     /codon_start=1
                     /gene="trxA"
                     /product="E. coli thioredoxin"
                     /label=TrxA
                     /translation="MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDE
                     IADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFL
                     DANLA"
     CDS             761..778
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             788..811
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             1279..1296
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1297..1341
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1422..1440)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1436..1483
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1520..1975
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1607..1626)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1817..1838
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2067..2882)
                     /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(2067..2882)
                     /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     2790..2809
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2874..2893
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3004..3592
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3493..3512
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3746..3763
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3778..3917
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3843..3862)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4003..4025
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4019..4210)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4782..5864)
                     /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     5826..5845
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5865..5942)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga
       61 tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc
      121 tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc
      181 cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcg
      241 agatcgatct cgatcccgcg aaattaatac gactcactat aggggaattg tgagcggata
      301 acaattcccc tctagaaata attttgttta actttaagaa ggagatatac atatgagcga
      361 taaaattatt cacctgactg acgacagttt tgacacggat gtactcaaag cggacggggc
      421 gatcctcgtc gatttctggg cagagtggtg cggtccgtgc aaaatgatcg ccccgattct
      481 ggatgaaatc gctgacgaat atcagggcaa actgaccgtt gcaaaactga acatcgatca
      541 aaaccctggc actgcgccga aatatggcat ccgtggtatc ccgactctgc tgctgttcaa
      601 aaacggtgaa gtggcggcaa ccaaagtggg tgcactgtct aaaggtcagt tgaaagagtt
      661 cctcgacgct aacctggccg gttctggttc tggccatact agtggtggtg gcggttctaa
      721 taacaatcct cctactccta ctccatctag tggttctggt catcaccatc accatcactc
      781 cgcggctctt gaagtcctct ttcagggacc cgggtaccag gatccgaatt cagattcttg
      841 cttggactta cacctcatgt cttctggagc tgcacgtgca atggtccatg cgtggctact
      901 aaacatacgc tccattgtct atgaaggtca tgagttacct aaagtcttga gtatattgac
      961 tggctggggg aaacacagca aagtggttgg agacggagca ctaagacgag cggttgaagt
     1021 tcttttgagg ggaatggatg caccattcca cctgtcaaaa tgcaacatgg gacggttcac
     1081 atcaagcggt tcggtggtag caacttggct gcgtgaatca gccacactga aactcttaat
     1141 cctccatgac cacataacaa ccgccacagc tactactaca acaatgaaat caacagacca
     1201 acaacaacgg aaacagacgt cgtttgcttt acagcctctt cttttgtagt cgacaagctt
     1261 gcggccgcag agctcgctct ggtgccacgc ggtagtaaag aaaccgctgc tgctaaattc
     1321 gaacgccagc acatggacag ctctacttct gctgctctcg aggcttaatt aacctaggct
     1381 gctaaacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc
     1441 ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat
     1501 atccggattg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     1561 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
     1621 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     1681 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
     1741 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     1801 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
     1861 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
     1921 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg
     1981 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     2041 atatgtatcc gctcatgaat taattcttag aaaaactcat cgagcatcaa atgaaactgc
     2101 aatttattca tatcaggatt atcaatacca tatttttgaa aaagccgttt ctgtaatgaa
     2161 ggagaaaact caccgaggca gttccatagg atggcaagat cctggtatcg gtctgcgatt
     2221 ccgactcgtc caacatcaat acaacctatt aatttcccct cgtcaaaaat aaggttatca
     2281 agtgagaaat caccatgagt gacgactgaa tccggtgaga atggcaaaag tttatgcatt
     2341 tctttccaga cttgttcaac aggccagcca ttacgctcgt catcaaaatc actcgcatca
     2401 accaaaccgt tattcattcg tgattgcgcc tgagcgagac gaaatacgcg atcactgtta
     2461 aaaggacaat tacaaacagg aatcgaatgc aaccggcgca ggaacactgc cagcgcatca
     2521 acaatatttt cacctgaatc aggatattct tctaatacct ggaatgctgt tttgccgggg
     2581 atcgcagtgg tgagtaacca tgcatcatca ggagtacgga taaaatgctt gatggtcgga
     2641 agaggcataa attccgtcag ccagtttagt ctgaccatct catctgtaac atcattggca
     2701 acgctacctt tgccatgttt cagaaacaac tctggcgcat cgggcttccc atacaatcga
     2761 tagattgtcg cacctgattg cccgacatta tcgcgagccc atttataccc atataaatca
     2821 gcatccatgt tggaatttaa tcgcggccta gagcaagacg tttcccgttg aatatggctc
     2881 ataacacccc ttgtattact gtttatgtaa gcagacagtt ttattgttca tgaccaaaat
     2941 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     3001 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     3061 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     3121 cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca
     3181 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     3241 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     3301 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     3361 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     3421 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     3481 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     3541 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     3601 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     3661 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
     3721 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct
     3781 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcaatg gtgcactctc
     3841 agtacaatct gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg
     3901 actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt
     3961 gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc
     4021 agaggttttc accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt
     4081 ggtcgtgaag cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct
     4141 ccagaagcgt taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct
     4201 gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga
     4261 tgaaacgaga gaggatgctc acgatacggg ttactgatga tgaacatgcc cggttactgg
     4321 aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga aaaatcactc
     4381 agggtcaatg ccagcgcttc gttaatacag atgtaggtgt tccacagggt agccagcagc
     4441 atcctgcgat gcagatccgg aacataatgg tgcagggcgc tgacttccgc gtttccagac
     4501 tttacgaaac acggaaaccg aagaccattc atgttgttgc tcaggtcgca gacgttttgc
     4561 agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc attctgctaa ccagtaaggc
     4621 aaccccgcca gcctagccgg gtcctcaacg acaggagcac gatcatgcta gtcatgcccc
     4681 gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatcccg
     4741 gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt
     4801 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     4861 tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg
     4921 cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc
     4981 aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta
     5041 tcgtcgtatc ccactaccga gatgtccgca ccaacgcgca gcccggactc ggtaatggcg
     5101 cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc
     5161 tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt
     5221 tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga
     5281 cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg
     5341 accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg
     5401 ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca
     5461 gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg
     5521 agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc
     5581 accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc
     5641 gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt
     5701 tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc
     5761 cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag
     5821 acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat
     5881 tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg
     5941 tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc
//
