LOCUS       sequence-208277-        5180 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 5180)
  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 5180)
  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-CPK30-Flag
FEATURES             Location/Qualifiers
     source          1..5180
                     /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             2148..2171
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2175..2198
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     terminator      2207..2459
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     primer_bind     complement(2498..2514)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2498..2514)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2511..2533)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2522..2538
                     /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(2546..2552,2553..2570,2571..2576))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2591..2612
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2729..2746)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2900..3488)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2980..2999)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3659..4450,4451..4519))
                     /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     4282..4301
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4520..4624)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4692..4710
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4748..4770)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4870..4889
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5083..5105
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5097..5114
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5098..5114
                     /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 atcgcctgcg
      541 tcaagtttga cccagacaac tcaaaaccaa accagaagaa gaaacctcca cggggacggc
      601 aacgaaaccc atacgacgat cccgatggtt taagaactca cgcaccgtta cgtgtgattc
      661 ctatgagcca tcaatctcag atcagcgaca agtacatctt aggacgagaa ctcggtcgcg
      721 gcgaattcgg aatcacgtat ctttgtacag atagagagac tcgtgaagct ttagcttgca
      781 aatcaatctc caagagaaag ctccgaaccg ccgtcgatgt ggaagacgtc cgtcgtgaag
      841 tcacgatcat gtcaacttta ccggaacacc caaacgttgt gaaacttaaa gcgacttatg
      901 aggataacga gaacgtgcat cttgtgatgg agctttgtga aggaggtgag ctttttgatc
      961 ggattgttgc cagaggacat tatacagagc gtgcggcggc taccgtcgcg agaacgatcg
     1021 cggaggttgt gaggatgtgt catgtcaatg gtgttatgca tagagatttg aagcctgaga
     1081 atttcttgtt tgctaacaag aaggagaatt ctgcacttaa ggctattgat tttggtttat
     1141 ctgttctctt taaacctgga gagaggttta cagagattgt tggaagtcct tattatatgg
     1201 ctccagaagt gttgaagaga aattatggac cagaggttga tgtgtggagt gctggagtta
     1261 tcctctacat cttgctttgt ggtgttcctc cgttttgggc agagactgaa caaggtgtgg
     1321 ctcttgccat cttgagggga gttcttgatt ttaagagaga tccttggtcg cagatatcag
     1381 agagcgcaaa gagccttgtg aagcagatgt tggaacctga ttcaactaag cgtttgactg
     1441 ctcagcaagt tcttgatcac ccttggatac agaatgcaaa gaaagctcca aatgttcctt
     1501 taggggatat tgttaggtca aggttgaagc aattctccat gatgaataga ttgaaaaaga
     1561 aagctctgcg tgttatcgcc gaacacttgt ctattcaaga agttgaagtg atcagaaaca
     1621 tgtttacgct gatggatgat gacaacgacg gtaaaatatc atatctagaa ctcagagctg
     1681 gactccggaa agtcggttca caactgggtg aaccagagat caaattgtta atggaagtgg
     1741 cggatgttaa tggaaatgga tgcttggact atggagagtt tgtagcagtg ataattcact
     1801 tgcagaagat ggagaatgat gagcatttca gacaagcttt tatgttcttt gacaaagatg
     1861 gaagtggata catcgaatca gaagaactac gtgaagcttt aaccgacgag ttaggtgaac
     1921 cagacaacag tgttataatc gatataatgc gtgaagtcga cactgacaag gacggaaaaa
     1981 taaactatga tgagtttgtg gtgatgatga aagctggaac tgattggaga aaggcatcaa
     2041 gacaatactc aagagagagg tttaaaagct taagcttaaa cttgatgaaa gacgggtcaa
     2101 tgcatctaca cgacgcactc actggacaat ctattgcagt taggcctgac tacaaggacg
     2161 acgatgacaa gggtgactac aaggacgacg atgacaagtg actgcagatc gttcaaacat
     2221 ttggcaataa agtttcttaa gattgaatcc tgttgccggt cttgcgatga ttatcatata
     2281 atttctgttg aattacgtta agcatgtaat aattaacatg taatgcatga cgttatttat
     2341 gagatgggtt tttatgatta gagtcccgca attatacatt taatacgcga tagaaaacaa
     2401 aatatagcgc gcaaactagg ataaattatc gcgcgcggtg tcatctatgt tactagatcc
     2461 gatgataagc tgtcaaacat gagaattcgt aatcatggtc atagctgttt cctgtgtgaa
     2521 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     2581 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     2641 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     2701 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     2761 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     2821 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     2881 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     2941 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     3001 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     3061 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     3121 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     3181 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     3241 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     3301 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     3361 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     3421 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     3481 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     3541 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     3601 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     3661 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     3721 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     3781 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     3841 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     3901 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     3961 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     4021 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     4081 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     4141 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     4201 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     4261 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     4321 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     4381 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     4441 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     4501 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     4561 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     4621 cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat
     4681 taacctataa aaataggcgt atcacgaggc cctttcgtct cgcgcgtttc ggtgatgacg
     4741 gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg
     4801 ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggctggc
     4861 ttaactatgc ggcatcagag cagattgtac tgagagtgca ccatatgcgg tgtgaaatac
     4921 cgcacagatg cgtaaggaga aaataccgca tcaggcgcca ttcgccattc aggctgcgca
     4981 actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg
     5041 gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta
     5101 aaacgacggc cagtgccaag ctctcgagaa gcttactcca agaatatcaa agatacagtc
     5161 tcagaagacc aaagggctat
//
