LOCUS       sequence-380014-        5241 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Nimble cloning-compatible vector for plant ectopic expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5241)
  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 5241)
  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-Green0029
FEATURES             Location/Qualifiers
     source          1..5241
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        181..283
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             314..619
                     /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     498..517
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     complement(718..738)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(718..736)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(757..773)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(757..773)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(770..792)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    781..797
                     /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(805..811,812..829,830..835))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    850..871
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(988..1005)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    1110..1134
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     rep_origin      1285..1720
                     /label=pSa ori
                     /note="origin of replication from bacterial plasmid pSa"
     primer_bind     complement(2000..2019)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2011..2826
                     /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             2011..2826
                     /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     complement(2084..2103)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2927..3515
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3416..3435
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    3582..3604
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA 
                     (truncated)"
     terminator      complement(3628..3880)
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     CDS             complement(3920..4714)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4031..4050)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4641..4660
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(4748..4927)
                     /label=NOS promoter
                     /note="nopaline synthase promoter"
     primer_bind     5159..5181
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5173..5190
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5174..5190
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5200..5219
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5200..5218
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 agtggtctct gtccagtcct ggcctcgtcg gccattctcg actaagttgg cagcatcacc
       61 cgacgcactt tgcgccgaat aaatacctgt gacggaagat cacttcgcag aataaataaa
      121 tcctggtgtc cctgttgata ccgggaagcc ctgggccaac ttttggcgaa aatgagacgt
      181 tgatcggcac gtaagaggtt ccaactttca ccataatgaa ataagatcac taccgggcgt
      241 attttttgag ttatcgagat tttcaggagc taaggaagct aaacttttgc tgacgagaac
      301 agggactggt gaaatgcagt ttaaggttta cacctataaa agagagagcc gttatcgtct
      361 gtttgtggat gtacagagtg atattattga cacgcctggg cgacggatgg tgatccccct
      421 ggccagtgca cgtctgctgt cagataaagt ctcccgtgaa ctttacccgg tggtgcatat
      481 cggggatgaa agctggcgca tgatgaccac cgatatggcc agtgtgccgg tatccgttat
      541 cggggaagaa gtggctgatc tcagccaccg cgaaaatgac atcaaaaacg ccattaacct
      601 gatgttctgg ggaatataaa tgtcaggctc ccttatacac agggccagtc tggccacttg
      661 tggtctgctg agacctctag agcggccgcc accgcggtgg agctccagct tttgttccct
      721 ttagtgaggg ttaattgcgc gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa
      781 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
      841 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
      901 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
      961 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     1021 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     1081 ggataacgca ggaaagaaca tgaaggcctt gacaggatat attggcgggt aaactaagtc
     1141 gctgtatgtg tttgtttgag atctaattcc ggggatcgga aatccagaag cccgagaggt
     1201 tgccgccttt cgggcttttt ctttttcaaa aaaaaaaatt tataaaacga tctgttgcgg
     1261 ccggccgccg ggttgtgggc aaaggcgctc gacggtgggc aaccgcttgc ggttgtccac
     1321 gggcggagcc ggtgcgcgta gcgcattgtc cacaagccaa gggcgaccaa taattgatat
     1381 atatattcat aattgaaaag ctaattgaac atactacttg ctgtaactac ttgccggagc
     1441 gaggggtgtt tgcaagctgt tgatctgaaa gggctattag cgttctcacg tgcctttttg
     1501 attagcgatt tcacgtgacc ttattagcga tttcacgtac tccgattagc gatttcacgt
     1561 accctgatta gcgatttcac gtggatagtt tttggagcgg gccggaaagc cccgtgaatc
     1621 aaggctttgc ggggcattag cggtttcacg tggataacta ccctctatcc acaggcttcc
     1681 ggggataaaa aagcccgctc gacggcgggc tgttggatgg ggatcgcctg aatcgcccca
     1741 tcatccagcc agaaagtgag ggagccacgg ttgatgagag ctttgttgta ggtggaccag
     1801 ttggtgattt tgaacttttg ctttgccacg gaacggtctg cgttgtcggg aagatgcgtg
     1861 atctgatcct tcaactcagc aaaagttcga tttattcaac aaagccacgt tgtggctcaa
     1921 aatctctgat gttacattgc acaagataaa aatatatcat catgaacaat aaaactgtct
     1981 gcttacataa acagtaatac aaggggtgtt atgagccata ttcaacggga aacgtcttgc
     2041 tcaaggccgc gattaaattc caacatggat gctgatttat atgggtataa atgggctcgc
     2101 gataatgtcg ggcaatcagg tgcgacaatc taccgattgt atgggaagcc cgatgcgcca
     2161 gagttgtttc tgaaacatgg caaaggtagc gttgccaatg atgttacaga tgagatggtc
     2221 agactaaact ggctgacgga atttatgcct cttccgacca tcaagcattt tatccgtact
     2281 cctgatgatg catggttact caccactgcg atcccaggga aaacagcatt ccaggtatta
     2341 gaagaatatc ctgattcagg tgaaaatatt gttgatgcgc tggcagtgtt cctgcgccgg
     2401 ttgcattcga ttcctgtttg taattgtcct tttaacagcg atcgcgtatt tcgtctcgct
     2461 caggcgcaat cacgaatgaa taacggtttg gttgatgcga gtgattttga tgacgagcgt
     2521 aatggctggc ctgttgaaca agtctggaaa gaaatgcata aacttttgcc attctcaccg
     2581 gattcagtcg tcactcatgg tgatttctca cttgataacc ttatttttga cgaggggaaa
     2641 ttaataggtt gtattgatgt tggacgagtc ggaatcgcag accgatacca ggatcttgcc
     2701 atcctatgga actgcctcgg tgagttttct ccttcattac agaaacggct ttttcaaaaa
     2761 tatggtattg ataatcctga tatgaataaa ttgcagtttc atttgatgct cgatgagttt
     2821 ttctaatcag aattggttaa ttggttgtaa ccatgaccaa aatcccttaa cgtgagtttt
     2881 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     2941 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     3001 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     3061 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     3121 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     3181 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     3241 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     3301 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     3361 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     3421 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     3481 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     3541 ggttcctggc cttttgctgg ccttttgctc acatgagatc ttggcaggat atattgtggt
     3601 gtaacgttat cagcttgcat gccggtcgat ctagtaacat agatgacacc gcgcgcgata
     3661 atttatccta gtttgcgcgc tatattttgt tttctatcgc gtattaaatg tataattgcg
     3721 ggactctaat cataaaaacc catctcataa ataacgtcat gcattacatg ttaattatta
     3781 catgcttaac gtaattcaac agaaattata tgataatcat cgcaagaccg gcaacaggat
     3841 tcaatcttaa gaaactttat tgccaaatgt ttgaacgatc tgcttgactc tagctagagt
     3901 ccgaacccca gagtcccgct cagaagaact cgtcaagaag gcgatagaag gcgatgcgct
     3961 gcgaatcggg agcggcgata ccgtaaagca cgaggaagcg gtcagcccat tcgccgccaa
     4021 gctcttcagc aatatcacgg gtagccaacg ctatgtcctg atagcggtcc gccacaccca
     4081 gccggccaca gtcgatgaat ccagaaaagc ggccattttc caccatgata ttcggcaagc
     4141 aggcatcgcc gtgggtcacg acgagatcct cgccgtcggg catccgcgcc ttgagcctgg
     4201 cgaacagttc ggctggcgcg agcccctgat gctcttcgtc cagatcatcc tgatcgacaa
     4261 gaccggcttc catccgagta cgtgctcgct cgatgcgatg tttcgcttgg tggtcgaatg
     4321 ggcaggtagc cggatcaagc gtatgcagcc gccgcattgc atcagccatg atggatactt
     4381 tctcggcagg agcaaggtga gatgacagga gatcctgccc cggcacttcg cccaatagca
     4441 gccagtccct tcccgcttca gtgacaacgt cgagcacagc tgcgcaagga acgcccgtcg
     4501 tggccagcca cgatagccgc gctgcctcgt cttggagttc attcagggca ccggacaggt
     4561 cggtcttgac aaaaagaacc gggcgcccct gcgctgacag ccggaacacg gcggcatcag
     4621 agcagccgat tgtctgttgt gcccagtcat agccgaatag cctctccacc caagcggccg
     4681 gagaacctgc gtgcaatcca tcttgttcaa tcatgcctcg atcgagttga gagtgaatat
     4741 gagactctaa ttggataccg aggggaattt atggaacgtc agtggagcat ttttgacaag
     4801 aaatatttgc tagctgatag tgaccttagg cgacttttga acgcgcaata atggtttctg
     4861 acgtatgtgc ttagctcatt aaactccaga aacccgcggc tcagtggctc cttcaacgtt
     4921 gcggttctgt cagttccaaa cgtaaaacgg cttgtcccgc gtcatcggcg ggggtcataa
     4981 cgtgactccc ttaattctcc gctcatgatc gataacatta acgcttacaa tttccattcg
     5041 ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc
     5101 cagctggcga aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc
     5161 cagtcacgac gttgtaaaac gacggccagt gagcgcgcgt aatacgactc actatagggc
     5221 gaattgggta ccgggccccc c
//
