LOCUS       sequence-208281-        5171 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 5171)
  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 5171)
  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-CPK32-Flag
FEATURES             Location/Qualifiers
     source          1..5171
                     /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             2139..2162
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2166..2189
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     terminator      2198..2450
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     primer_bind     complement(2489..2505)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2489..2505)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2502..2524)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2513..2529
                     /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(2537..2543,2544..2561,2562..2567))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2582..2603
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2720..2737)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2891..3479)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2971..2990)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3650..4441,4442..4510))
                     /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     4273..4292
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4511..4615)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4683..4701
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4739..4761)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4861..4880
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5074..5096
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5088..5105
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5089..5105
                     /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 gggtaattgt tgcggaacag
      541 caggatcatt ggctcaaaac gataacaaac ccaaaaaagg aaggaagaag caaaaccctt
      601 tctcgattga ttacggtctt caccacggtg gtggagatgg aggtggaaga cctcttaagc
      661 ttatcgtttt gaatgatcca acaggtcgtg agatcgagtc taaatacacg ttggggagag
      721 agctaggtcg tggagaattc ggtgttacgt atctatgtac tgataaggag acagacgacg
      781 tttttgcttg taaatcgatt ttgaagaaga agctgaggac agctgttgat attgaagatg
      841 ttaggagaga ggttgagatt atgaggcata tgcctgagca tcctaatgtt gttactttga
      901 aggagactta tgaggatgag catgctgttc atttggttat ggagctttgt gaaggtggtg
      961 aattgtttga taggattgtt gctagagggc attatactga gagagctgct gctgctgtca
     1021 ctaagaccat catggaagtt gttcaggtgt gtcataagca tggggtaatg cacagggacc
     1081 tgaaacctga gaacttcttg tttggaaaca agaaggagac tgcacctctt aaggcgattg
     1141 attttggtct ctctgttttc tttaaaccag gcgagaggtt taacgaaatc gttggtagtc
     1201 cgtactacat ggctcccgag gtgctaaaac ggaattatgg tccagaagtt gatatttgga
     1261 gtgcaggtgt aattctttac atactgctat gtggtgtccc gcctttctgg gcagaaactg
     1321 aacaaggagt tgcacaagca attattcgat ctgtactaga cttcagaagg gacccatggc
     1381 ccaaggtttc tgaaaacgca aaagacctta tcaggaaaat gcttgatcct gaccaaaagc
     1441 gtcgtcttac agctcaacaa gtcctagatc acccgtggtt acagaatgca aagacagccc
     1501 ccaatgtatc attgggtgaa acagtgaggg caaggttgaa gcagttcacc gtcatgaaca
     1561 agctcaagaa acgagcactc agggttattg ctgagcattt atcagatgaa gaagcttcag
     1621 gtataagaga agggttccaa ataatggaca caagccaaag agggaagatt aacatcgacg
     1681 agcttaaaat cgggttgcag aaacttggtc atgccattcc gcaagatgat ttacaaattt
     1741 tgatggatgc tggagacatc gatagagatg gatatttgga ctgtgacgag tttattgcca
     1801 tatcggtaca cctaaggaaa atgggcaatg acgagcatct caagaaagcg tttgcgttct
     1861 ttgatcaaaa caataacgga tatatcgaaa tagaggagtt aagggaagcc ttgtctgacg
     1921 aacttggtac aagtgaagaa gttgttgatg ccattatccg tgatgttgac accgacaagg
     1981 atggaagaat aagttacgaa gagtttgtaa cgatgatgaa aacaggaaca gattggagaa
     2041 aagcttcgag acagtactcg agagaacggt ttaacagtat cagtctcaaa ctgatgcaag
     2101 atgcgtcgtt gcaggtcaat ggtgatacaa gaaggcctga ctacaaggac gacgatgaca
     2161 agggtgacta caaggacgac gatgacaagt gactgcagat cgttcaaaca tttggcaata
     2221 aagtttctta agattgaatc ctgttgccgg tcttgcgatg attatcatat aatttctgtt
     2281 gaattacgtt aagcatgtaa taattaacat gtaatgcatg acgttattta tgagatgggt
     2341 ttttatgatt agagtcccgc aattatacat ttaatacgcg atagaaaaca aaatatagcg
     2401 cgcaaactag gataaattat cgcgcgcggt gtcatctatg ttactagatc cgatgataag
     2461 ctgtcaaaca tgagaattcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     2521 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     2581 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     2641 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     2701 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     2761 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     2821 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     2881 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     2941 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     3001 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     3061 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     3121 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     3181 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     3241 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     3301 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     3361 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     3421 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     3481 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     3541 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     3601 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     3661 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     3721 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     3781 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     3841 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     3901 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     3961 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     4021 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     4081 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     4141 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     4201 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     4261 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     4321 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     4381 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     4441 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     4501 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     4561 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     4621 ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata
     4681 aaaataggcg tatcacgagg ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc
     4741 tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca
     4801 gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg cttaactatg
     4861 cggcatcaga gcagattgta ctgagagtgc accatatgcg gtgtgaaata ccgcacagat
     4921 gcgtaaggag aaaataccgc atcaggcgcc attcgccatt caggctgcgc aactgttggg
     4981 aagggcgatc ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg
     5041 caaggcgatt aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg
     5101 ccagtgccaa gctctcgaga agcttactcc aagaatatca aagatacagt ctcagaagac
     5161 caaagggcta t
//
