LOCUS       sequence-208273-        4538 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For Arabidopsis mesophyll protoplast transient expression assay.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4538)
  AUTHORS   Liu KH, Niu Y, Konishi M, Wu Y, Du H, Sun Chung H, Li L, Boudsocq M,
            McCormack M, Maekawa S, Ishida T, Zhang C, Shokat K, Yanagisawa S, 
            Sheen J
  TITLE     Discovery of nitrate-CPK-NLP signalling in central nutrient-growth 
            networks.
  JOURNAL   Nature. 2017 May 18;545(7654):311-316. doi: 10.1038/nature22077. 
            Epub 2017 May 10.
  PUBMED    28489820
REFERENCE   2  (bases 1 to 4538)
  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/nature22077"; journalName: "Nature"; date: "2017-05-18- 
            18"; volume: "545"; issue: "7654"; pages: "311-316"
COMMENT     Sequence Label: HBT-CPK10ac-Flag
FEATURES             Location/Qualifiers
     source          1..4538
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(1..299,300..322,323..510)
                     /label=35SPPDK promoter
                     /note="hybrid promoter consisting of the cauliflower mosaic
                     virus 35S enhancer fused to the maize C4PPDK basal promoter
                     (Yoo et al., 2007)"
     CDS             1506..1529
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1533..1556
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     terminator      1565..1817
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     primer_bind     complement(1856..1872)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1856..1872)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1869..1891)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1880..1896
                     /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)."
     promoter        complement(join(1904..1910,1911..1928,1929..1934))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1949..1970
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2087..2104)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2258..2846)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2338..2357)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3017..3808,3809..3877))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     3640..3659
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3878..3982)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4050..4068
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4106..4128)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4228..4247
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4441..4463
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4455..4472
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4456..4472
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 tgagactttt caacaaaggg taatatcggg aaacctcctc ggattccatt gcccagctat
       61 ctgtcacttc atcaaaagga cagtagaaaa ggaaggtggc acctacaaat gccatcattg
      121 cgataaagga aaggctatcg ttcaagatgc ctctgccgac agtggtccca aagatggacc
      181 cccacccaca aggagcatcg tggaaaaaga agacgttcca accacgtctt caaagcaagt
      241 ggattgatgt gatatctcca ctgacgtaag ggatgacgca caatcccact atccttcgcc
      301 ccaagcttgg gcccaagctt gggtcgcgcc ccacggatgg tataagaata aaggcattcc
      361 gcgtgcagga ttcacccgtt cgcctctcac cttttcgctg tactctctcg ccacacacac
      421 cccctctcca gctccgttgg agctccggac agcagcaggc gcggggcggt cacgtagtaa
      481 gcagctctcg gctccctctc cccttgctcc gtggatccat gggtaactgt aacgcctgtg
      541 taaggccaga ctcaaaagaa tcaaaaccat cttcaaagcc gaaaaaaccc aatcgagatc
      601 ggaaattaaa cccattcgcc ggagatttca ccagatcccc agctccaata cgtgttctca
      661 aagatgtaat ccctatgagc aatcaaactc agatcagcga caaatacatc ttaggtcgtg
      721 aattaggtcg aggcgaattc ggaatcactt acctctgtac tgatcgtgaa acccacgaag
      781 ctttagcttg caaatcgatt tcaaagcgaa agcttcgaac agctgtcgat atcgaagacg
      841 ttcgtcgtga ggtagcgatt atgtctactt tacctgagca tccaaacgta gttaagctta
      901 aggctagtta tgaggataac gagaacgtgc atctggttat ggagctttgt gaaggaggtg
      961 agctttttga tcggattgtt gctagaggac attacacgga gcgtgctgct gcagctgttg
     1021 cgagaacgat tgctgaggtt gtgatgatgt gtcactctaa tggagttatg catcgagatt
     1081 tgaaacctga gaatttcttg tttgctaata aaaaggagaa ttctccacta aaggctattg
     1141 attttggctt gtctgtgttc ttcaaacctg gagataagtt tacagagatt gtaggaagtc
     1201 cgtattatat ggctccagaa gtgttgaaga gagattatgg accaggggtt gatgtgtgga
     1261 gtgccggagt tattatctat atcttgctct gtggtgttcc tccgttttgg gctgagactg
     1321 aacaaggtgt tgctcttgcg atcttgcggg gagttcttga ttttaagaga gacccttggc
     1381 ctcagatatc agagagtgcc aagagccttg tgaagcagat gttggatcct gatccgacta
     1441 agcggttaac tgctcagcaa gtgttagctc acccatggat acagaatgca aagaaaggta
     1501 ggcctgacta caaggacgac gatgacaagg gtgactacaa ggacgacgat gacaagtgac
     1561 tgcagatcgt tcaaacattt ggcaataaag tttcttaaga ttgaatcctg ttgccggtct
     1621 tgcgatgatt atcatataat ttctgttgaa ttacgttaag catgtaataa ttaacatgta
     1681 atgcatgacg ttatttatga gatgggtttt tatgattaga gtcccgcaat tatacattta
     1741 atacgcgata gaaaacaaaa tatagcgcgc aaactaggat aaattatcgc gcgcggtgtc
     1801 atctatgtta ctagatccga tgataagctg tcaaacatga gaattcgtaa tcatggtcat
     1861 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     1921 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     1981 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     2041 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     2101 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     2161 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     2221 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     2281 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     2341 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     2401 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     2461 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     2521 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     2581 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     2641 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     2701 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     2761 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     2821 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     2881 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     2941 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     3001 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     3061 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     3121 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     3181 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     3241 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     3301 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     3361 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     3421 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     3481 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     3541 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     3601 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     3661 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     3721 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     3781 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     3841 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     3901 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     3961 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa
     4021 ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtctcg
     4081 cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag
     4141 cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg
     4201 gcgggtgtcg gggctggctt aactatgcgg catcagagca gattgtactg agagtgcacc
     4261 atatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggcgccatt
     4321 cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac
     4381 gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt
     4441 cccagtcacg acgttgtaaa acgacggcca gtgccaagct ctcgagaagc ttactccaag
     4501 aatatcaaag atacagtctc agaagaccaa agggctat
//
