LOCUS       sequence-208271-        5195 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 5195)
  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 5195)
  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-CPK10-Flag
FEATURES             Location/Qualifiers
     source          1..5195
                     /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             2163..2186
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2190..2213
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     terminator      2222..2474
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     primer_bind     complement(2513..2529)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2513..2529)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2526..2548)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2537..2553
                     /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(2561..2567,2568..2585,2586..2591))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2606..2627
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2744..2761)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2915..3503)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2995..3014)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3674..4465,4466..4534))
                     /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     4297..4316
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4535..4639)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4707..4725
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4763..4785)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4885..4904
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5098..5120
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5112..5129
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5113..5129
                     /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 aagaaagctc
     1501 ccaatgttcc tttaggagat atagtcagat ctaggttgaa gcagttctct atgatgaaca
     1561 gattcaaaaa gaaagttctt cgtgtaattg cggagcactt gtctattcaa gaggttgaag
     1621 tgataaagaa catgttctca ctgatggatg atgacaagga tggtaaaata acttacccgg
     1681 aactcaaagc tgggcttcag aaggtcggtt cacaacttgg tgaaccagag atcaaaatgt
     1741 tgatggaagt ggcggatgtc gatggaaatg ggtttctgga ttatggagag tttgtagctg
     1801 tgataattca cttgcagaag atagagaatg atgaactttt caaactagct tttatgtttt
     1861 tcgacaaaga tggaagtaca tacattgaac ttgatgagct acgggaagct ttagcggatg
     1921 agttaggcga gccagacgcc agtgttctaa gcgacatcat gcgtgaagtt gacactgaca
     1981 aggacggacg tataaactat gatgagtttg tgacgatgat gaaagctgga actgactgga
     2041 gaaaggcatc gagacaatat tcaagagaga ggttcaaaag cttaagcatt aacttgatga
     2101 aagatgggtc attgcatctc catgacgctc tcactggaca aactgttcct gttggtaggc
     2161 ctgactacaa ggacgacgat gacaagggtg actacaagga cgacgatgac aagtgactgc
     2221 agatcgttca aacatttggc aataaagttt cttaagattg aatcctgttg ccggtcttgc
     2281 gatgattatc atataatttc tgttgaatta cgttaagcat gtaataatta acatgtaatg
     2341 catgacgtta tttatgagat gggtttttat gattagagtc ccgcaattat acatttaata
     2401 cgcgatagaa aacaaaatat agcgcgcaaa ctaggataaa ttatcgcgcg cggtgtcatc
     2461 tatgttacta gatccgatga taagctgtca aacatgagaa ttcgtaatca tggtcatagc
     2521 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     2581 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     2641 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     2701 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     2761 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     2821 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     2881 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     2941 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     3001 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     3061 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     3121 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     3181 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     3241 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     3301 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     3361 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     3421 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     3481 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     3541 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     3601 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     3661 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     3721 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     3781 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     3841 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     3901 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     3961 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     4021 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     4081 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     4141 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     4201 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     4261 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     4321 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     4381 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     4441 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     4501 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     4561 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     4621 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca
     4681 ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtctcgcgc
     4741 gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt
     4801 gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg
     4861 ggtgtcgggg ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccata
     4921 tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg cgccattcgc
     4981 cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc
     5041 agctggcgaa agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc
     5101 agtcacgacg ttgtaaaacg acggccagtg ccaagctctc gagaagctta ctccaagaat
     5161 atcaaagata cagtctcaga agaccaaagg gctat
//
