LOCUS       sequence-380178-        5487 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 5487)
  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 5487)
  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-C
FEATURES             Location/Qualifiers
     source          1..5487
                     /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..1931
                     /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(1366..1384)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1634..1653
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     terminator      1954..2206
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     primer_bind     complement(2224..2240)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2224..2240)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(2277..2297)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2277..2295)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2316..2332)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2316..2332)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2329..2351)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2340..2356
                     /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(2364..2370,2371..2388,2389..2394))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2409..2430
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2547..2564)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2669..2693
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     rep_origin      complement(2784..3372)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2864..2883)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3543..4358)
                     /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(3543..4358)
                     /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     4266..4285
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4350..4369
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4649..5084
                     /label=pSa ori
                     /note="origin of replication from bacterial plasmid pSa"
     misc_feature    5211..5233
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA 
                     (truncated)"
     primer_bind     5383..5405
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5397..5414
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5398..5414
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5424..5443
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5424..5442
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     5467..5483
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     5468..5484
                     /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 atggtgagca agggcgagga ggataacatg gccatcatca
     1261 aggagttcat gcgcttcaag gtgcacatgg agggctccgt gaacggccac gagttcgaga
     1321 tcgagggcga gggcgagggc cgcccctacg agggcaccca gaccgccaag ctgaaggtga
     1381 ccaagggtgg ccccctgccc ttcgcctggg acatcctgtc ccctcagttc atgtacggct
     1441 ccaaggccta cgtgaagcac cccgccgaca tccccgacta cttgaagctg tccttccccg
     1501 agggcttcaa gtgggagcgc gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc
     1561 aggactcctc cctgcaggac ggcgagttca tctacaaggt gaagctgcgc ggcaccaact
     1621 tcccctccga cggccccgta atgcagaaga agaccatggg ctgggaggcc tcctccgagc
     1681 ggatgtaccc cgaggacggc gccctgaagg gcgagatcaa gcagaggctg aagctgaagg
     1741 acggcggcca ctacgacgct gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc
     1801 tgcccggcgc ctacaacgtc aacatcaagt tggacatcac ctcccacaac gaggactaca
     1861 ccatcgtgga acagtacgaa cgcgccgagg gccgccactc caccggcggc atggacgagc
     1921 tgtacaagtg aggtgaccag ctcgaatttc cccgatcgtt caaacatttg gcaataaagt
     1981 ttcttaagat tgaatcctgt tgccggtctt gcgatgatta tcatataatt tctgttgaat
     2041 tacgttaagc atgtaataat taacatgtaa tgcatgacgt tatttatgag atgggttttt
     2101 atgattagag tcccgcaatt atacatttaa tacgcgatag aaaacaaaat atagcgcgca
     2161 aactaggata aattatcgcg cgcggtgtca tctatgttac tagatcgggc ctgcagcccg
     2221 ggggatccac tagttctaga gcggccgcca ccgcggtgga gctccagctt ttgttccctt
     2281 tagtgagggt taattgcgcg cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     2341 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     2401 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     2461 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     2521 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     2581 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     2641 gataacgcag gaaagaacat gaaggccttg acaggatata ttggcgggta aactaagtcg
     2701 ctgtatgtgt ttgtttgaga tctcatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     2761 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     2821 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     2881 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     2941 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     3001 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     3061 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     3121 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     3181 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     3241 tgcgctctgc tgaagccagt taccttcgga agaagagttg gtagctcttg atccggcaaa
     3301 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     3361 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     3421 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     3481 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgtgtaaca ttggtctagt
     3541 gattagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca
     3601 ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc
     3661 cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa
     3721 cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg
     3781 actgaatccg gtgagaatgg caaaagttta tgcatttctt tccagacttg ttcaacaggc
     3841 cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt cattcgtgat
     3901 tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca aacaggaatc
     3961 gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tattttcacc tgaatcagga
     4021 tattcttcta atacctggaa tgctgttttc cctgggatcg cagtggtgag taaccatgca
     4081 tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag
     4141 tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga
     4201 aacaactctg gcgcatcggg cttcccatac aatcggtaga ttgtcgcacc tgattgcccg
     4261 acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga atttaatcgc
     4321 ggccttgagc aagacgtttc ccgttgaata tggctcataa caccccttgt attactgttt
     4381 atgtaagcag acagttttat tgttcatgat gatatatttt tatcttgtgc aatgtaacat
     4441 cagagatttt gagacacaac gtggctttgt tgaataaatc gaacttttgc tgagttgaag
     4501 gatcagatca cgcatcttcc cgacaacgca gaccgttccg tggcaaagca aaagttcaaa
     4561 atcaccaact ggtccaccta caacaaagct ctcatcaacc gtggctccct cactttctgg
     4621 ctggatgatg gggcgattca ggcgatcccc atccaacagc ccgccgtcga gcgggctttt
     4681 ttatccccgg aagcctgtgg atagagggta gttatccacg tgaaaccgct aatgccccgc
     4741 aaagccttga ttcacggggc tttccggccc gctccaaaaa ctatccacgt gaaatcgcta
     4801 atcagggtac gtgaaatcgc taatcggagt acgtgaaatc gctaataagg tcacgtgaaa
     4861 tcgctaatca aaaaggcacg tgagaacgct aatagccctt tcagatcaac agcttgcaaa
     4921 cacccctcgc tccggcaagt agttacagca agtagtatgt tcaattagct tttcaattat
     4981 gaatatatat atcaattatt ggtcgccctt ggcttgtgga caatgcgcta cgcgcaccgg
     5041 ctccgcccgt ggacaaccgc aagcggttgc ccaccgtcga gcgcctttgc ccacaacccg
     5101 gcggccggcc gcaacagatc gttttataaa tttttttttt tgaaaaagaa aaagcccgaa
     5161 aggcggcaac ctctcgggct tctggatttc cgatccccgg aattagatct tggcaggata
     5221 tattgtggtg taacgttaac attaacgctt acaatttcca ttcgccattc aggctgcgca
     5281 actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg
     5341 gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta
     5401 aaacgacggc cagtgagcgc gcgtaatacg actcactata gggcgaattg ggtaccgggc
     5461 cccccctcga ggtcgacggt atcgata
//
