LOCUS       sequence-449178-        6166 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the Salicylic acid binding core (SBC) of NPR4 in E.coli, 
            in an insoluble form.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6166)
  AUTHORS   Wang W, Withers J, Li H, Zwack PJ, Rusnac DV, Shi H, Liu L, Yan S, 
            Hinds TR, Guttman M, Dong X, Zheng N
  TITLE     Structural basis of salicylic acid perception by Arabidopsis NPR 
            proteins.
  JOURNAL   Nature. 2020 Oct;586(7828):311-316. doi: 10.1038/s41586-020-2596-y. 
            Epub 2020 Aug 12.
  PUBMED    32788727
REFERENCE   2  (bases 1 to 6166)
  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: 
            "10.1038/s41586-020-2596-y"; journalName: "Nature"; date: "2020-10";
            volume: "586"; issue: "7828"; pages: "311-316"
COMMENT     Sequence Label: pET-NPR4 (SA binding domain)
FEATURES             Location/Qualifiers
     source          1..6166
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     51..73
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(67..258)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     2613..2632
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2652..2729)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2935..2954
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3038..3057
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3038..3056
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3057..3081
                     /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             3096..3118
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3137..3154
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3164..3181
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             3188..3208
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(3705..3723)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3719..3766
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(3941..3946,3947..3963,3964..3969))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(3996..4014)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4082..4186
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4187..4255,4256..5047)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4405..4424)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5218..5806
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5707..5726
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5960..5977
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5992..6131
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6057..6076)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
       61 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc
      121 atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt
      181 gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt
      241 tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat
      301 gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg
      361 gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa
      421 aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag
      481 ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt
      541 ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga
      601 cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc
      661 agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgcgcac
      721 ccgtggccag gacccaacgc tgcccgagat gcgccgcgtg cggctgctgg agatggcgga
      781 cgcgatggat atgttctgcc aagggttggt ttgcgcattc acagttctcc gcaagaattg
      841 attggctcca attcttggag tggtgaatcc gttagcgagg tgccgccggc ttccattcag
      901 gtcgaggtgg cccggctcca tgcaccgcga cgcaacgcgg ggaggcagac aaggtatagg
      961 gcggcgccta caatccatgc caacccgttc catgtgctcg ccgaggcggc ataaatcgcc
     1021 gtgacgatca gcggtccaat gatcgaagtt aggctggtaa gagccgcgag cgatccttga
     1081 agctgtccct gatggtcgtc atctacctgc ctggacagca tggcctgcaa cgcgggcatc
     1141 ccgatgccgc cggaagcgag aagaatcata atggggaagg ccatccagcc tcgcgtcgcg
     1201 aacgccagca agacgtagcc cagcgcgtcg gccgccatgc cggcgataat ggcctgcttc
     1261 tcgccgaaac gtttggtggc gggaccagtg acgaaggctt gagcgagggc gtgcaagatt
     1321 ccgaataccg caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg
     1381 aaaatgaccc agagcgctgc cggcacctgt cctacgagtt gcatgataaa gaagacagtc
     1441 ataagtgcgg cgacgatagt catgccccgc gcccaccgga aggagctgac tgggttgaag
     1501 gctctcaagg gcatcggtcg agatcccggt gcctaatgag tgagctaact tacattaatt
     1561 gcgttgcgct cactgcccgc tttccagtcg ggaaactgtc gtgccagctg cattaatgaa
     1621 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc
     1681 accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc
     1741 aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc
     1801 gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat atccgcacca
     1861 acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca
     1921 accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg
     1981 gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga
     2041 tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac
     2101 agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct
     2161 tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc
     2221 ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta
     2281 atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg
     2341 acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat
     2401 ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca
     2461 atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc
     2521 tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc
     2581 accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt
     2641 actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg
     2701 cgaaaggttt tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc
     2761 ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa
     2821 tggtgcatgc aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc
     2881 cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat
     2941 gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg
     3001 tccggcgtag aggatcgaga tctcgatccc gcgaaattaa tacgactcac tataggggaa
     3061 ttgtgagcgg ataacaattc ccctctagaa ataattttgt ttaactttaa gaaggagata
     3121 taccatgggc agcagccatc atcatcatca tcacagcagc ggcctggtgc cgcgcggcag
     3181 ccatagcgaa aacctctact tccaatcgat ggaacctagt aaataccgct tatgcatcga
     3241 tatcttggaa agggaaatta gaaggaatcc attggttagt ggggatacac ccacttgttc
     3301 ccattcgatg cccgaggatc tccaaatgag gttgttatac ttagaaaagc gagtgggact
     3361 tgctcagttg ttcttcccag cagaagccaa tgtggctatg gacgttgcta atgttgaagg
     3421 gacaagcgag tgcacaggtc ttaatgatac aactgaaaac ttgggtaaag tcgatttaaa
     3481 tgaaacgcct tatgtgcaaa cgaaaagaat gcttacacgt atgaaagccc tcatgaaaac
     3541 agttgagaca ggtcggagat acttcccatc ttgttatgag gttctggata agtacatgga
     3601 tcagtatatg gacgaagaaa tcccttaata accggaggat gagtgtggcc tcgaggatcc
     3661 ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact
     3721 agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac
     3781 tatatccgga tatcccgcaa gaggcccggc agtaccggca taaccaagcc tatgcctaca
     3841 gcatccaggg tgacggtgcc gaggatgacg atgagcgcat tgttagattt catacacggt
     3901 gcctgactgc gttagcaatt taactgtgat aaactaccgc attaaagctt atcgatgata
     3961 agctgtcaaa catgagaatt cttgaagacg aaagggcctc gtgatacgcc tatttttata
     4021 ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt
     4081 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     4141 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     4201 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     4261 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     4321 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     4381 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgtg ttgacgccgg
     4441 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     4501 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     4561 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     4621 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     4681 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg cagcaatggc
     4741 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     4801 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     4861 tggctggttt attgctgata aatctggagc cggtgagcgt ggctctcgcg gtatcattgc
     4921 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     4981 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     5041 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     5101 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     5161 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     5221 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     5281 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     5341 cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa
     5401 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     5461 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     5521 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     5581 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     5641 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     5701 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     5761 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     5821 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     5881 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     5941 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg
     6001 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca
     6061 atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg
     6121 tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctg
//
