LOCUS       sequence-380181-        5454 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Nimble cloning-compatible vector for protein subcellular 
            localization in plants.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5454)
  AUTHORS   Yan P, Tuo D, Shen W, Deng H, Zhou P, Gao X
  TITLE     A Nimble Cloning-compatible vector system for high-throughput gene 
            functional analysis in plants.
  JOURNAL   Plant Commun. 2023 Mar 13;4(2):100471. doi: 
            10.1016/j.xplc.2022.100471. Epub 2022 Nov 9.
  PUBMED    36352791
REFERENCE   2  (bases 1 to 5454)
  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"; journalName: "Plant 
            Commun."; date: "2023-03-13"; volume: "4(2):100471. doi"; pages: " 
            10.1016/j.xplc.2022.100471. Epub 2022 Nov 9"
COMMENT     Sequence Label: pNC-RFP-C
FEATURES             Location/Qualifiers
     source          1..5454
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        184..529
                     /label=CaMV 35S promoter
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     primer_bind     472..492
                     /label=35S promoter
                     /note="CaMV 35S promoter, forward primer"
     promoter        725..827
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             858..1163
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     1042..1061
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             1221..1898
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     CDS             1221..1898
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /note="mammalian codon-optimized"
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     primer_bind     complement(1351..1369)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1619..1638
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1813..1836
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     terminator      1921..2173
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     primer_bind     complement(2191..2207)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2191..2207)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(2244..2264)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2244..2262)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2283..2299)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2283..2299)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2296..2318)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2307..2323
                     /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(2331..2337,2338..2355,2356..2361))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2376..2397
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2514..2531)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2636..2660
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     rep_origin      complement(2751..3339)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2831..2850)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3510..4325)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(3510..4325)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     4233..4252
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4317..4336
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4616..5051
                     /label=pSa ori
                     /note="origin of replication from bacterial plasmid pSa"
     misc_feature    5178..5200
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA 
                     (truncated)"
     primer_bind     5350..5372
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5364..5381
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5365..5381
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5391..5410
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5391..5409
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     5434..5450
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     5435..5451
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 catggagtca aagattcaaa tagaggacct aacagaactc gccgtaaaga ctggcgaaca
       61 gttcatacag agtctcttac gactcaatga caagaagaaa atcttcgtca acatggtgga
      121 gcacgacaca cttgtctact ccaaaaatat caaagataca gtctcagaag accaaagggc
      181 aattgagact tttcaacaaa gggtaatatc cggaaacctc ctcggattcc attgcccagc
      241 tatctgtcac tttattgtga agatagtgga aaaggaaggt ggctcctaca aatgccatca
      301 ttgcgataaa ggaaaggcca tcgttgaaga tgcctctgcc gacagtggtc ccaaagatgg
      361 acccccaccc acgaggagca tcgtggaaaa agaagacgtt ccaaccacgt cttcaaagca
      421 agtggattga tgtgatatct ccactgacgt aagggatgac gcacaatccc actatccttc
      481 gcaagaccct tcctctatat aaggaagttc atttcatttg gagagaacac gggggactct
      541 tgacagtggt ctctgtccag tcctggcctc gtcggccatt ctcgactaag ttggcagcat
      601 cacccgacgc actttgcgcc gaataaatac ctgtgacgga agatcacttc gcagaataaa
      661 taaatcctgg tgtccctgtt gataccggga agccctgggc caacttttgg cgaaaatgag
      721 acgttgatcg gcacgtaaga ggttccaact ttcaccataa tgaaataaga tcactaccgg
      781 gcgtattttt tgagttatcg agattttcag gagctaagga agctaaactt ttgctgacga
      841 gaacagggac tggtgaaatg cagtttaagg tttacaccta taaaagagag agccgttatc
      901 gtctgtttgt ggatgtacag agtgatatta ttgacacgcc tgggcgacgg atggtgatcc
      961 ccctggccag tgcacgtctg ctgtcagata aagtctcccg tgaactttac ccggtggtgc
     1021 atatcgggga tgaaagctgg cgcatgatga ccaccgatat ggccagtgtg ccggtatccg
     1081 ttatcgggga agaagtggct gatctcagcc accgcgaaaa tgacatcaaa aacgccatta
     1141 acctgatgtt ctggggaata taaatgtcag gctcccttat acacagggcc agtctggcca
     1201 cttgtggtct gctgagaccg atggcctcct ccgaggacgt catcaaggag ttcatgcgct
     1261 tcaaggtgcg catggagggc tccgtgaacg gccacgagtt cgagatcgag ggcgagggcg
     1321 agggccgccc ctacgagggc acccagaccg ccaagctgaa ggtgaccaag ggcggccccc
     1381 tgcccttcgc ctgggacatc ctgtcccctc agttccagta cggctccaag gcctacgtga
     1441 agcaccccgc cgacatcccc gactacttga agctgtcctt ccccgagggc ttcaagtggg
     1501 agcgcgtgat gaacttcgag gacggcggcg tggtgaccgt gacccaggac tcctccctgc
     1561 aggacggcga gttcatctac aaggtgaagc tgcgcggcac caacttcccc tccgacggcc
     1621 ccgtaatgca gaagaagacc atgggctggg aggcctccac cgagcggatg taccccgagg
     1681 acggcgccct gaagggcgag atcaagatga ggctgaagct gaaggacggc ggccactacg
     1741 acgccgaggt caagaccacc tacatggcca agaagcccgt gcagctgccc ggcgcctaca
     1801 agaccgacat caagctggac atcacctccc acaacgagga ctacaccatc gtggaacagt
     1861 acgagcgcgc cgagggccgc cactccaccg gcgcctgagg tgaccagctc gaatttcccc
     1921 gatcgttcaa acatttggca ataaagtttc ttaagattga atcctgttgc cggtcttgcg
     1981 atgattatca tataatttct gttgaattac gttaagcatg taataattaa catgtaatgc
     2041 atgacgttat ttatgagatg ggtttttatg attagagtcc cgcaattata catttaatac
     2101 gcgatagaaa acaaaatata gcgcgcaaac taggataaat tatcgcgcgc ggtgtcatct
     2161 atgttactag atcgggcctg cagcccgggg gatccactag ttctagagcg gccgccaccg
     2221 cggtggagct ccagcttttg ttccctttag tgagggttaa ttgcgcgctt ggcgtaatca
     2281 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     2341 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     2401 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     2461 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     2521 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     2581 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgaa ggccttgaca
     2641 ggatatattg gcgggtaaac taagtcgctg tatgtgtttg tttgagatct catgtgagca
     2701 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     2761 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     2821 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     2881 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     2941 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     3001 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     3061 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     3121 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     3181 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaga
     3241 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     3301 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     3361 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     3421 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     3481 agtatatatg tgtaacattg gtctagtgat tagaaaaact catcgagcat caaatgaaac
     3541 tgcaatttat tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat
     3601 gaaggagaaa actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg
     3661 attccgactc gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta
     3721 tcaagtgaga aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc
     3781 atttctttcc agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca
     3841 tcaaccaaac cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg
     3901 ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca
     3961 tcaacaatat tttcacctga atcaggatat tcttctaata cctggaatgc tgttttccct
     4021 gggatcgcag tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc
     4081 ggaagaggca taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg
     4141 gcaacgctac ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat
     4201 cggtagattg tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa
     4261 tcagcatcca tgttggaatt taatcgcggc cttgagcaag acgtttcccg ttgaatatgg
     4321 ctcataacac cccttgtatt actgtttatg taagcagaca gttttattgt tcatgatgat
     4381 atatttttat cttgtgcaat gtaacatcag agattttgag acacaacgtg gctttgttga
     4441 ataaatcgaa cttttgctga gttgaaggat cagatcacgc atcttcccga caacgcagac
     4501 cgttccgtgg caaagcaaaa gttcaaaatc accaactggt ccacctacaa caaagctctc
     4561 atcaaccgtg gctccctcac tttctggctg gatgatgggg cgattcaggc gatccccatc
     4621 caacagcccg ccgtcgagcg ggctttttta tccccggaag cctgtggata gagggtagtt
     4681 atccacgtga aaccgctaat gccccgcaaa gccttgattc acggggcttt ccggcccgct
     4741 ccaaaaacta tccacgtgaa atcgctaatc agggtacgtg aaatcgctaa tcggagtacg
     4801 tgaaatcgct aataaggtca cgtgaaatcg ctaatcaaaa aggcacgtga gaacgctaat
     4861 agccctttca gatcaacagc ttgcaaacac ccctcgctcc ggcaagtagt tacagcaagt
     4921 agtatgttca attagctttt caattatgaa tatatatatc aattattggt cgcccttggc
     4981 ttgtggacaa tgcgctacgc gcaccggctc cgcccgtgga caaccgcaag cggttgccca
     5041 ccgtcgagcg cctttgccca caacccggcg gccggccgca acagatcgtt ttataaattt
     5101 ttttttttga aaaagaaaaa gcccgaaagg cggcaacctc tcgggcttct ggatttccga
     5161 tccccggaat tagatcttgg caggatatat tgtggtgtaa cgttaacatt aacgcttaca
     5221 atttccattc gccattcagg ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt
     5281 cgctattacg ccagctggcg aaagggggat gtgctgcaag gcgattaagt tgggtaacgc
     5341 cagggttttc ccagtcacga cgttgtaaaa cgacggccag tgagcgcgcg taatacgact
     5401 cactataggg cgaattgggt accgggcccc ccctcgaggt cgacggtatc gata
//
