LOCUS       sequence-208279-        4547 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 4547)
  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 4547)
  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-CPK30ac-Flag
FEATURES             Location/Qualifiers
     source          1..4547
                     /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             1512..1535
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     terminator      1544..1796
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     primer_bind     complement(1835..1851)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1835..1851)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1848..1870)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1859..1875
                     /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(1883..1889,1890..1907,1908..1913))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1928..1949
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2066..2083)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2237..2825)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2317..2336)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2996..3787,3788..3856))
                     /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     3619..3638
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3857..3961)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4029..4047
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4085..4107)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4207..4226
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4420..4442
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4434..4451
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4435..4451
                     /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 cccacccacg aggagcatcg tggaaaaaga agacgttcca accacgtctt caaagcaagt
      241 ggattgatgt gatatctcca ctgacgtaag ggatgacgca caatcccact atccttcgcc
      301 ccaagcttgg gcccaagctt gggtcgcgcc ccacggatgg tataagaata aaggcattcc
      361 gcgtgcagga ttcacccgtt cgcctctcac cttttcgctg tactcactcg ccacacacac
      421 cccctctcca gctccgttgg agctccggac agcagcaggc gcggggcggt cacgtagtaa
      481 gcagctctcg gctccctctc cccttgctcc atatgatcgt gcaacccatc ggatccatgg
      541 gtaattgtat cgcctgcgtc aagtttgacc cagacaactc aaaaccaaac cagaagaaga
      601 aacctccacg gggacggcaa cgaaacccat acgacgatcc cgatggttta agaactcacg
      661 caccgttacg tgtgattcct atgagccatc aatctcagat cagcgacaag tacatcttag
      721 gacgagaact cggtcgcggc gaattcggaa tcacgtatct ttgtacagat agagagactc
      781 gtgaagcttt agcttgcaaa tcaatctcca agagaaagct ccgaaccgcc gtcgatgtgg
      841 aagacgtccg tcgtgaagtc acgatcatgt caactttacc ggaacaccca aacgttgtga
      901 aacttaaagc gacttatgag gataacgaga acgtgcatct tgtgatggag ctttgtgaag
      961 gaggtgagct ttttgatcgg attgttgcca gaggacatta tacagagcgt gcggcggcta
     1021 ccgtcgcgag aacgatcgcg gaggttgtga ggatgtgtca tgtcaatggt gttatgcata
     1081 gagatttgaa gcctgagaat ttcttgtttg ctaacaagaa ggagaattct gcacttaagg
     1141 ctattgattt tggtttatct gttctcttta aacctggaga gaggtttaca gagattgttg
     1201 gaagtcctta ttatatggct ccagaagtgt tgaagagaaa ttatggacca gaggttgatg
     1261 tgtggagtgc tggagttatc ctctacatct tgctttgtgg tgttcctccg ttttgggcag
     1321 agactgaaca aggtgtggct cttgccatct tgaggggagt tcttgatttt aagagagatc
     1381 cttggtcgca gatatcagag agcgcaaaga gccttgtgaa gcagatgttg gaacctgatt
     1441 caactaagcg tttgactgct cagcaagttc ttgatcaccc ttggatacag aatgcaaaga
     1501 aaaggccttc tgactacaag gacgacgatg acaagtgact gcagatcgtt caaacatttg
     1561 gcaataaagt ttcttaagat tgaatcctgt tgccggtctt gcgatgatta tcatataatt
     1621 tctgttgaat tacgttaagc atgtaataat taacatgtaa tgcatgacgt tatttatgag
     1681 atgggttttt atgattagag tcccgcaatt atacatttaa tacgcgatag aaaacaaaat
     1741 atagcgcgca aactaggata aattatcgcg cgcggtgtca tctatgttac tagatccgat
     1801 gataagctgt caaacatgag aattcgtaat catggtcata gctgtttcct gtgtgaaatt
     1861 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     1921 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     1981 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     2041 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     2101 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     2161 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     2221 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     2281 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     2341 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     2401 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     2461 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     2521 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     2581 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     2641 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     2701 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     2761 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     2821 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     2881 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     2941 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     3001 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     3061 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     3121 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     3181 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     3241 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     3301 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     3361 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     3421 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     3481 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     3541 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     3601 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     3661 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     3721 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     3781 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     3841 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     3901 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     3961 gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa
     4021 cctataaaaa taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg
     4081 aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg
     4141 ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta
     4201 actatgcggc atcagagcag attgtactga gagtgcacca tatgcggtgt gaaataccgc
     4261 acagatgcgt aaggagaaaa taccgcatca ggcgccattc gccattcagg ctgcgcaact
     4321 gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg aaagggggat
     4381 gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa
     4441 cgacggccag tgccaagctt ctagagatcc gtcaacatgg tggagcacga cactctcgtc
     4501 tactccaaga atatcaaaga tacagtctca gaagaccaaa gggctat
//
