LOCUS       sequence-380180-        5488 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 5488)
  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 5488)
  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-mCherry-N
FEATURES             Location/Qualifiers
     source          1..5488
                     /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"
     CDS             545..1252
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(690..708)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     958..977
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     promoter        1434..1536
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             1567..1872
                     /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     1751..1770
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     terminator      1955..2207
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     primer_bind     complement(2225..2241)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2225..2241)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(2278..2298)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2278..2296)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2317..2333)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2317..2333)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2330..2352)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2341..2357
                     /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(2365..2371,2372..2389,2390..2395))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2410..2431
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2548..2565)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2670..2694
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     rep_origin      complement(2785..3373)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2865..2884)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3544..4359)
                     /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(3544..4359)
                     /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     4267..4286
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4351..4370
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4650..5085
                     /label=pSa ori
                     /note="origin of replication from bacterial plasmid pSa"
     misc_feature    5212..5234
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA 
                     (truncated)"
     primer_bind     5384..5406
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5398..5415
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5399..5415
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5425..5444
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5425..5443
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     5468..5484
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     5469..5485
                     /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 tgacatggtg agcaagggcg aggaggataa catggccatc atcaaggagt tcatgcgctt
      601 caaggtgcac atggagggct ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga
      661 gggccgcccc tacgagggca cccagaccgc caagctgaag gtgaccaagg gtggccccct
      721 gcccttcgcc tgggacatcc tgtcccctca gttcatgtac ggctccaagg cctacgtgaa
      781 gcaccccgcc gacatccccg actacttgaa gctgtccttc cccgagggct tcaagtggga
      841 gcgcgtgatg aacttcgagg acggcggcgt ggtgaccgtg acccaggact cctccctgca
      901 ggacggcgag ttcatctaca aggtgaagct gcgcggcacc aacttcccct ccgacggccc
      961 cgtaatgcag aagaagacca tgggctggga ggcctcctcc gagcggatgt accccgagga
     1021 cggcgccctg aagggcgaga tcaagcagag gctgaagctg aaggacggcg gccactacga
     1081 cgctgaggtc aagaccacct acaaggccaa gaagcccgtg cagctgcccg gcgcctacaa
     1141 cgtcaacatc aagttggaca tcacctccca caacgaggac tacaccatcg tggaacagta
     1201 cgaacgcgcc gagggccgcc actccaccgg cggcatggac gagctgtaca agcagtggtc
     1261 tctgtccagt cctggcctcg tcggccattc tcgactaagt tggcagcatc acccgacgca
     1321 ctttgcgccg aataaatacc tgtgacggaa gatcacttcg cagaataaat aaatcctggt
     1381 gtccctgttg ataccgggaa gccctgggcc aacttttggc gaaaatgaga cgttgatcgg
     1441 cacgtaagag gttccaactt tcaccataat gaaataagat cactaccggg cgtatttttt
     1501 gagttatcga gattttcagg agctaaggaa gctaaacttt tgctgacgag aacagggact
     1561 ggtgaaatgc agtttaaggt ttacacctat aaaagagaga gccgttatcg tctgtttgtg
     1621 gatgtacaga gtgatattat tgacacgcct gggcgacgga tggtgatccc cctggccagt
     1681 gcacgtctgc tgtcagataa agtctcccgt gaactttacc cggtggtgca tatcggggat
     1741 gaaagctggc gcatgatgac caccgatatg gccagtgtgc cggtatccgt tatcggggaa
     1801 gaagtggctg atctcagcca ccgcgaaaat gacatcaaaa acgccattaa cctgatgttc
     1861 tggggaatat aaatgtcagg ctcccttata cacagggcca gtctggccac ttgtggtctg
     1921 ctgagaccgt gaggtgacca gctcgaattt ccccgatcgt tcaaacattt ggcaataaag
     1981 tttcttaaga ttgaatcctg ttgccggtct tgcgatgatt atcatataat ttctgttgaa
     2041 ttacgttaag catgtaataa ttaacatgta atgcatgacg ttatttatga gatgggtttt
     2101 tatgattaga gtcccgcaat tatacattta atacgcgata gaaaacaaaa tatagcgcgc
     2161 aaactaggat aaattatcgc gcgcggtgtc atctatgtta ctagatcggg cctgcagccc
     2221 gggggatcca ctagttctag agcggccgcc accgcggtgg agctccagct tttgttccct
     2281 ttagtgaggg ttaattgcgc gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa
     2341 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
     2401 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
     2461 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     2521 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     2581 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     2641 ggataacgca ggaaagaaca tgaaggcctt gacaggatat attggcgggt aaactaagtc
     2701 gctgtatgtg tttgtttgag atctcatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     2761 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     2821 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     2881 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     2941 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     3001 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     3061 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     3121 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     3181 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     3241 ctgcgctctg ctgaagccag ttaccttcgg aagaagagtt ggtagctctt gatccggcaa
     3301 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     3361 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     3421 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     3481 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgtgtaac attggtctag
     3541 tgattagaaa aactcatcga gcatcaaatg aaactgcaat ttattcatat caggattatc
     3601 aataccatat ttttgaaaaa gccgtttctg taatgaagga gaaaactcac cgaggcagtt
     3661 ccataggatg gcaagatcct ggtatcggtc tgcgattccg actcgtccaa catcaataca
     3721 acctattaat ttcccctcgt caaaaataag gttatcaagt gagaaatcac catgagtgac
     3781 gactgaatcc ggtgagaatg gcaaaagttt atgcatttct ttccagactt gttcaacagg
     3841 ccagccatta cgctcgtcat caaaatcact cgcatcaacc aaaccgttat tcattcgtga
     3901 ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa ggacaattac aaacaggaat
     3961 cgaatgcaac cggcgcagga acactgccag cgcatcaaca atattttcac ctgaatcagg
     4021 atattcttct aatacctgga atgctgtttt ccctgggatc gcagtggtga gtaaccatgc
     4081 atcatcagga gtacggataa aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca
     4141 gtttagtctg accatctcat ctgtaacatc attggcaacg ctacctttgc catgtttcag
     4201 aaacaactct ggcgcatcgg gcttcccata caatcggtag attgtcgcac ctgattgccc
     4261 gacattatcg cgagcccatt tatacccata taaatcagca tccatgttgg aatttaatcg
     4321 cggccttgag caagacgttt cccgttgaat atggctcata acaccccttg tattactgtt
     4381 tatgtaagca gacagtttta ttgttcatga tgatatattt ttatcttgtg caatgtaaca
     4441 tcagagattt tgagacacaa cgtggctttg ttgaataaat cgaacttttg ctgagttgaa
     4501 ggatcagatc acgcatcttc ccgacaacgc agaccgttcc gtggcaaagc aaaagttcaa
     4561 aatcaccaac tggtccacct acaacaaagc tctcatcaac cgtggctccc tcactttctg
     4621 gctggatgat ggggcgattc aggcgatccc catccaacag cccgccgtcg agcgggcttt
     4681 tttatccccg gaagcctgtg gatagagggt agttatccac gtgaaaccgc taatgccccg
     4741 caaagccttg attcacgggg ctttccggcc cgctccaaaa actatccacg tgaaatcgct
     4801 aatcagggta cgtgaaatcg ctaatcggag tacgtgaaat cgctaataag gtcacgtgaa
     4861 atcgctaatc aaaaaggcac gtgagaacgc taatagccct ttcagatcaa cagcttgcaa
     4921 acacccctcg ctccggcaag tagttacagc aagtagtatg ttcaattagc ttttcaatta
     4981 tgaatatata tatcaattat tggtcgccct tggcttgtgg acaatgcgct acgcgcaccg
     5041 gctccgcccg tggacaaccg caagcggttg cccaccgtcg agcgcctttg cccacaaccc
     5101 ggcggccggc cgcaacagat cgttttataa attttttttt ttgaaaaaga aaaagcccga
     5161 aaggcggcaa cctctcgggc ttctggattt ccgatccccg gaattagatc ttggcaggat
     5221 atattgtggt gtaacgttaa cattaacgct tacaatttcc attcgccatt caggctgcgc
     5281 aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg
     5341 ggatgtgctg caaggcgatt aagttgggta acgccagggt tttcccagtc acgacgttgt
     5401 aaaacgacgg ccagtgagcg cgcgtaatac gactcactat agggcgaatt gggtaccggg
     5461 ccccccctcg aggtcgacgg tatcgata
//
