LOCUS       sequence-208275-        4559 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 4559)
  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 4559)
  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-CPK32ac-Flag
FEATURES             Location/Qualifiers
     source          1..4559
                     /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             1524..1547
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     terminator      1556..1808
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     primer_bind     complement(1847..1863)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1847..1863)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1860..1882)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1871..1887
                     /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(1895..1901,1902..1919,1920..1925))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1940..1961
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2078..2095)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2249..2837)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2329..2348)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3008..3799,3800..3868))
                     /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     3631..3650
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3869..3973)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4041..4059
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4097..4119)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4219..4238
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4432..4454
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4446..4463
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4447..4463
                     /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 gtaattgttg cggaacagca ggatcattgg ctcaaaacga taacaaaccc aaaaaaggaa
      601 ggaagaagca aaaccctttc tcgattgatt acggtcttca ccacggtggt ggagatggag
      661 gtggaagacc tcttaagctt atcgttttga atgatccaac aggtcgtgag atcgagtcta
      721 aatacacgtt ggggagagag ctaggtcgtg gagaattcgg tgttacgtat ctatgtactg
      781 ataaggagac agacgacgtt tttgcttgta aatcgatttt gaagaagaag ctgaggacag
      841 ctgttgatat tgaagatgtt aggagagagg ttgagattat gaggcatatg cctgagcatc
      901 ctaatgttgt tactttgaag gagacttatg aggatgagca tgctgttcat ttggttatgg
      961 agctttgtga aggtggtgaa ttgtttgata ggattgttgc tagagggcat tatactgaga
     1021 gagctgctgc tgctgtcact aagaccatca tggaagttgt tcaggtgtgt cataagcatg
     1081 gggtaatgca cagggacctg aaacctgaga acttcttgtt tggaaacaag aaggagactg
     1141 cacctcttaa ggcgattgat tttggtctct ctgttttctt taaaccaggc gagaggttta
     1201 acgaaatcgt tggtagtccg tactacatgg ctcccgaggt gctaaaacgg aattatggtc
     1261 cagaagttga tatttggagt gcaggtgtaa ttctttacat actgctatgt ggtgtcccgc
     1321 ctttctgggc agaaactgaa caaggagttg cacaagcaat tattcgatct gtactagact
     1381 tcagaaggga cccatggccc aaggtttctg aaaacgcaaa agaccttatc aggaaaatgc
     1441 ttgatcctga ccaaaagcgt cgtcttacag ctcaacaagt cctagatcac ccgtggttac
     1501 agaatgcaaa gacaaggcct tctgactaca aggacgacga tgacaagtga ctgcagatcg
     1561 ttcaaacatt tggcaataaa gtttcttaag attgaatcct gttgccggtc ttgcgatgat
     1621 tatcatataa tttctgttga attacgttaa gcatgtaata attaacatgt aatgcatgac
     1681 gttatttatg agatgggttt ttatgattag agtcccgcaa ttatacattt aatacgcgat
     1741 agaaaacaaa atatagcgcg caaactagga taaattatcg cgcgcggtgt catctatgtt
     1801 actagatccg atgataagct gtcaaacatg agaattcgta atcatggtca tagctgtttc
     1861 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     1921 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     1981 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     2041 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     2101 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     2161 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     2221 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     2281 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     2341 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     2401 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     2461 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     2521 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     2581 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     2641 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     2701 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     2761 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     2821 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     2881 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     2941 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     3001 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     3061 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     3121 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     3181 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     3241 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     3301 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     3361 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     3421 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     3481 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     3541 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     3601 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     3661 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     3721 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     3781 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     3841 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     3901 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     3961 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accattatta
     4021 tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtctc gcgcgtttcg
     4081 gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt
     4141 aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc
     4201 ggggctggct taactatgcg gcatcagagc agattgtact gagagtgcac catatgcggt
     4261 gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggcgccat tcgccattca
     4321 ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg
     4381 cgaaaggggg atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac
     4441 gacgttgtaa aacgacggcc agtgccaagc ttctagagat ccgtcaacat ggtggagcac
     4501 gacactctcg tctactccaa gaatatcaaa gatacagtct cagaagacca aagggctat
//
