LOCUS       sequence-383230-        6119 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 6119)
  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 6119)
  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-Green0029-35S
FEATURES             Location/Qualifiers
     source          1..6119
                     /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"
     terminator      1251..1503
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     primer_bind     complement(1521..1537)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1521..1537)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(1574..1594)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1574..1592)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1613..1629)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1613..1629)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1626..1648)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1637..1653
                     /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(1661..1667,1668..1685,1686..1691))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1706..1727
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1844..1861)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    1966..1990
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     rep_origin      2141..2576
                     /label=pSa ori
                     /note="origin of replication from bacterial plasmid pSa"
     primer_bind     complement(2856..2875)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2867..3682
                     /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             2867..3682
                     /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(2940..2959)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3783..4371
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4272..4291
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    4438..4460
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA 
                     (truncated)"
     terminator      complement(4484..4736)
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     CDS             complement(4776..5570)
                     /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(4887..4906)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     5497..5516
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(5604..5783)
                     /label=NOS promoter
                     /note="nopaline synthase promoter"
     primer_bind     6015..6037
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6029..6046
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6030..6046
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6056..6075
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6056..6074
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     6099..6115
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     6100..6116
                     /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 tgtgaattgg tgaccagctc gaatttcccc gatcgttcaa
     1261 acatttggca ataaagtttc ttaagattga atcctgttgc cggtcttgcg atgattatca
     1321 tataatttct gttgaattac gttaagcatg taataattaa catgtaatgc atgacgttat
     1381 ttatgagatg ggtttttatg attagagtcc cgcaattata catttaatac gcgatagaaa
     1441 acaaaatata gcgcgcaaac taggataaat tatcgcgcgc ggtgtcatct atgttactag
     1501 atcgggcctg cagcccgggg gatccactag ttctagagcg gccgccaccg cggtggagct
     1561 ccagcttttg ttccctttag tgagggttaa ttgcgcgctt ggcgtaatca tggtcatagc
     1621 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     1681 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     1741 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     1801 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     1861 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     1921 tatccacaga atcaggggat aacgcaggaa agaacatgaa ggccttgaca ggatatattg
     1981 gcgggtaaac taagtcgctg tatgtgtttg tttgagatct aattccgggg atcggaaatc
     2041 cagaagcccg agaggttgcc gcctttcggg ctttttcttt ttcaaaaaaa aaaatttata
     2101 aaacgatctg ttgcggccgg ccgccgggtt gtgggcaaag gcgctcgacg gtgggcaacc
     2161 gcttgcggtt gtccacgggc ggagccggtg cgcgtagcgc attgtccaca agccaagggc
     2221 gaccaataat tgatatatat attcataatt gaaaagctaa ttgaacatac tacttgctgt
     2281 aactacttgc cggagcgagg ggtgtttgca agctgttgat ctgaaagggc tattagcgtt
     2341 ctcacgtgcc tttttgatta gcgatttcac gtgaccttat tagcgatttc acgtactccg
     2401 attagcgatt tcacgtaccc tgattagcga tttcacgtgg atagtttttg gagcgggccg
     2461 gaaagccccg tgaatcaagg ctttgcgggg cattagcggt ttcacgtgga taactaccct
     2521 ctatccacag gcttccgggg ataaaaaagc ccgctcgacg gcgggctgtt ggatggggat
     2581 cgcctgaatc gccccatcat ccagccagaa agtgagggag ccacggttga tgagagcttt
     2641 gttgtaggtg gaccagttgg tgattttgaa cttttgcttt gccacggaac ggtctgcgtt
     2701 gtcgggaaga tgcgtgatct gatccttcaa ctcagcaaaa gttcgattta ttcaacaaag
     2761 ccacgttgtg gctcaaaatc tctgatgtta cattgcacaa gataaaaata tatcatcatg
     2821 aacaataaaa ctgtctgctt acataaacag taatacaagg ggtgttatga gccatattca
     2881 acgggaaacg tcttgctcaa ggccgcgatt aaattccaac atggatgctg atttatatgg
     2941 gtataaatgg gctcgcgata atgtcgggca atcaggtgcg acaatctacc gattgtatgg
     3001 gaagcccgat gcgccagagt tgtttctgaa acatggcaaa ggtagcgttg ccaatgatgt
     3061 tacagatgag atggtcagac taaactggct gacggaattt atgcctcttc cgaccatcaa
     3121 gcattttatc cgtactcctg atgatgcatg gttactcacc actgcgatcc cagggaaaac
     3181 agcattccag gtattagaag aatatcctga ttcaggtgaa aatattgttg atgcgctggc
     3241 agtgttcctg cgccggttgc attcgattcc tgtttgtaat tgtcctttta acagcgatcg
     3301 cgtatttcgt ctcgctcagg cgcaatcacg aatgaataac ggtttggttg atgcgagtga
     3361 ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc tggaaagaaa tgcataaact
     3421 tttgccattc tcaccggatt cagtcgtcac tcatggtgat ttctcacttg ataaccttat
     3481 ttttgacgag gggaaattaa taggttgtat tgatgttgga cgagtcggaa tcgcagaccg
     3541 ataccaggat cttgccatcc tatggaactg cctcggtgag ttttctcctt cattacagaa
     3601 acggcttttt caaaaatatg gtattgataa tcctgatatg aataaattgc agtttcattt
     3661 gatgctcgat gagtttttct aatcagaatt ggttaattgg ttgtaaccat gaccaaaatc
     3721 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     3781 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     3841 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     3901 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     3961 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     4021 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     4081 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     4141 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     4201 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     4261 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     4321 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     4381 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gagatcttgg
     4441 caggatatat tgtggtgtaa cgttatcagc ttgcatgccg gtcgatctag taacatagat
     4501 gacaccgcgc gcgataattt atcctagttt gcgcgctata ttttgttttc tatcgcgtat
     4561 taaatgtata attgcgggac tctaatcata aaaacccatc tcataaataa cgtcatgcat
     4621 tacatgttaa ttattacatg cttaacgtaa ttcaacagaa attatatgat aatcatcgca
     4681 agaccggcaa caggattcaa tcttaagaaa ctttattgcc aaatgtttga acgatctgct
     4741 tgactctagc tagagtccga accccagagt cccgctcaga agaactcgtc aagaaggcga
     4801 tagaaggcga tgcgctgcga atcgggagcg gcgataccgt aaagcacgag gaagcggtca
     4861 gcccattcgc cgccaagctc ttcagcaata tcacgggtag ccaacgctat gtcctgatag
     4921 cggtccgcca cacccagccg gccacagtcg atgaatccag aaaagcggcc attttccacc
     4981 atgatattcg gcaagcaggc atcgccgtgg gtcacgacga gatcctcgcc gtcgggcatc
     5041 cgcgccttga gcctggcgaa cagttcggct ggcgcgagcc cctgatgctc ttcgtccaga
     5101 tcatcctgat cgacaagacc ggcttccatc cgagtacgtg ctcgctcgat gcgatgtttc
     5161 gcttggtggt cgaatgggca ggtagccgga tcaagcgtat gcagccgccg cattgcatca
     5221 gccatgatgg atactttctc ggcaggagca aggtgagatg acaggagatc ctgccccggc
     5281 acttcgccca atagcagcca gtcccttccc gcttcagtga caacgtcgag cacagctgcg
     5341 caaggaacgc ccgtcgtggc cagccacgat agccgcgctg cctcgtcttg gagttcattc
     5401 agggcaccgg acaggtcggt cttgacaaaa agaaccgggc gcccctgcgc tgacagccgg
     5461 aacacggcgg catcagagca gccgattgtc tgttgtgccc agtcatagcc gaatagcctc
     5521 tccacccaag cggccggaga acctgcgtgc aatccatctt gttcaatcat gcctcgatcg
     5581 agttgagagt gaatatgaga ctctaattgg ataccgaggg gaatttatgg aacgtcagtg
     5641 gagcattttt gacaagaaat atttgctagc tgatagtgac cttaggcgac ttttgaacgc
     5701 gcaataatgg tttctgacgt atgtgcttag ctcattaaac tccagaaacc cgcggctcag
     5761 tggctccttc aacgttgcgg ttctgtcagt tccaaacgta aaacggcttg tcccgcgtca
     5821 tcggcggggg tcataacgtg actcccttaa ttctccgctc atgatcgata acattaacgc
     5881 ttacaatttc cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc
     5941 ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt
     6001 aacgccaggg ttttcccagt cacgacgttg taaaacgacg gccagtgagc gcgcgtaata
     6061 cgactcacta tagggcgaat tgggtaccgg gccccccctc gaggtcgacg gtatcgata
//
