LOCUS       sequence-380048-        6980 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Nimble cloning-compatible vector for plant BiFC.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6980)
  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 6980)
  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-BiFC-Enc
FEATURES             Location/Qualifiers
     source          1..6980
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     10..26
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        445..789
                     /label=CaMV 35S promoter
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     primer_bind     733..753
                     /label=35S promoter
                     /note="CaMV 35S promoter, forward primer"
     promoter        1112..1214
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             1245..1550
                     /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     1429..1448
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             join(1608..1610,1611..1613,1614..2126)
                     /codon_start=1
                     /product="N-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VN173
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIE"
     primer_bind     complement(1653..1674)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1914..1933)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(2383..2399)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2383..2399)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(2436..2456)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2436..2454)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2475..2491)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2475..2491)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2488..2510)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2499..2515
                     /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(2523..2529,2530..2547,2548..2553))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2568..2589
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2706..2723)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2828..2852
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     rep_origin      3003..3438
                     /label=pSa ori
                     /note="origin of replication from bacterial plasmid pSa"
     primer_bind     complement(3718..3737)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3729..4544
                     /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             3729..4544
                     /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(3802..3821)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      4645..5233
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5134..5153
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    5300..5322
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA 
                     (truncated)"
     terminator      complement(5346..5598)
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     CDS             complement(5638..6432)
                     /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(5749..5768)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     6359..6378
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(6466..6645)
                     /label=NOS promoter
                     /note="nopaline synthase promoter"
     primer_bind     6877..6899
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6892..6908
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6918..6937
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6918..6936
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     6961..6977
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     6962..6978
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 aagcttgttg taaaacgacg gccagtgccg gcgcgccacc ggtcaacatg tggagcacga
       61 cacacttgtc tactccaaaa atatcaaaga tacagtctca gaagaccaaa gggcaattga
      121 gacttttcaa caaagggtaa tatccggaaa cctcctcgga ttccattgcc cagctatctg
      181 tcactttatt gtgaagatag tggaaaagga aggtggctcc tacaaatgcc atcattgcga
      241 taaaggaaag gccatcgttg aagatgcctc tgccgacagt ggtcccaaag atggaccccc
      301 acccacgagg agcatcgtgg aaaaagaaga cgttccaacc acgtcttcaa agcaagtgga
      361 ttgatgtgat aacatggtgg agcacgacac acttgtctac tccaaaaata tcaaagatac
      421 agtctcagaa gaccaaaggg caattgagac ttttcaacaa agggtaatat ccggaaacct
      481 cctcggattc cattgcccag ctatctgtca ctttattgtg aagatagtgg aaaaggaagg
      541 tggctcctac aaatgccatc attgcgataa aggaaaggcc atcgttgaag atgcctctgc
      601 cgacagtggt cccaaagatg gacccccacc cacgaggagc atcgtggaaa aagaagacgt
      661 tccaaccacg tcttcaaagc aagtggattg atgtgatatc tccactgacg taagggatga
      721 cgcacaatcc cactatcctt cgcaagaccc ttcctctata taaggaagtt catttcattt
      781 ggagaggacg tcgagagttc tcaacacaac atatacaaaa caaacgaatc tcaagcaatc
      841 aagcattcta cttctattgc agcaatttaa atcatttctt ttaaagcaaa agcaattttc
      901 tgaaaatttt caccatttac gaacgatagc cagtggtctc tgtccagtcc tggcctcgtc
      961 ggccattctc gactaagttg gcagcatcac ccgacgcact ttgcgccgaa taaatacctg
     1021 tgacggaaga tcacttcgca gaataaataa atcctggtgt ccctgttgat accgggaagc
     1081 cctgggccaa cttttggcga aaatgagacg ttgatcggca cgtaagaggt tccaactttc
     1141 accataatga aataagatca ctaccgggcg tattttttga gttatcgaga ttttcaggag
     1201 ctaaggaagc taaacttttg ctgacgagaa cagggactgg tgaaatgcag tttaaggttt
     1261 acacctataa aagagagagc cgttatcgtc tgtttgtgga tgtacagagt gatattattg
     1321 acacgcctgg gcgacggatg gtgatccccc tggccagtgc acgtctgctg tcagataaag
     1381 tctcccgtga actttacccg gtggtgcata tcggggatga aagctggcgc atgatgacca
     1441 ccgatatggc cagtgtgccg gtatccgtta tcggggaaga agtggctgat ctcagccacc
     1501 gcgaaaatga catcaaaaac gccattaacc tgatgttctg gggaatataa atgtcaggct
     1561 cccttataca cagggccagt ctggccactt gtggtctgct gagacccatg gtgagcaagg
     1621 gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg
     1681 gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc
     1741 tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccacct
     1801 tcggctacgg cctgcagtgc ttcgcccgct accccgacca catgaagcag cacgacttct
     1861 tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg
     1921 gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg
     1981 agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca
     2041 actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga
     2101 acttcaagat ccgccacaac atcgaggact gaagatccac ctgatctaga gtccgcaaaa
     2161 atcaccagtc tctctctaca aatctatctc tctctatttt tctccagaat aatgtgtgag
     2221 tagttcccag ataagggaat tagggttctt atagggtttc gctcatgtgt tgagcatata
     2281 agaaaccctt agtatgtatt tgtatttgta aaatacttct atcaataaaa tttctaattc
     2341 ctaaaaccaa aatccagtga cgcgaattcc tgcagcccgg gggatccact agttctagag
     2401 cggccgccac cgcggtggag ctccagcttt tgttcccttt agtgagggtt aattgcgcgc
     2461 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     2521 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     2581 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     2641 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     2701 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     2761 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     2821 aaggccttga caggatatat tggcgggtaa actaagtcgc tgtatgtgtt tgtttgagat
     2881 ctaattccgg ggatcggaaa tccagaagcc cgagaggttg ccgcctttcg ggctttttct
     2941 ttttcaaaaa aaaaaattta taaaacgatc tgttgcggcc ggccgccggg ttgtgggcaa
     3001 aggcgctcga cggtgggcaa ccgcttgcgg ttgtccacgg gcggagccgg tgcgcgtagc
     3061 gcattgtcca caagccaagg gcgaccaata attgatatat atattcataa ttgaaaagct
     3121 aattgaacat actacttgct gtaactactt gccggagcga ggggtgtttg caagctgttg
     3181 atctgaaagg gctattagcg ttctcacgtg cctttttgat tagcgatttc acgtgacctt
     3241 attagcgatt tcacgtactc cgattagcga tttcacgtac cctgattagc gatttcacgt
     3301 ggatagtttt tggagcgggc cggaaagccc cgtgaatcaa ggctttgcgg ggcattagcg
     3361 gtttcacgtg gataactacc ctctatccac aggcttccgg ggataaaaaa gcccgctcga
     3421 cggcgggctg ttggatgggg atcgcctgaa tcgccccatc atccagccag aaagtgaggg
     3481 agccacggtt gatgagagct ttgttgtagg tggaccagtt ggtgattttg aacttttgct
     3541 ttgccacgga acggtctgcg ttgtcgggaa gatgcgtgat ctgatccttc aactcagcaa
     3601 aagttcgatt tattcaacaa agccacgttg tggctcaaaa tctctgatgt tacattgcac
     3661 aagataaaaa tatatcatca tgaacaataa aactgtctgc ttacataaac agtaatacaa
     3721 ggggtgttat gagccatatt caacgggaaa cgtcttgctc aaggccgcga ttaaattcca
     3781 acatggatgc tgatttatat gggtataaat gggctcgcga taatgtcggg caatcaggtg
     3841 cgacaatcta ccgattgtat gggaagcccg atgcgccaga gttgtttctg aaacatggca
     3901 aaggtagcgt tgccaatgat gttacagatg agatggtcag actaaactgg ctgacggaat
     3961 ttatgcctct tccgaccatc aagcatttta tccgtactcc tgatgatgca tggttactca
     4021 ccactgcgat cccagggaaa acagcattcc aggtattaga agaatatcct gattcaggtg
     4081 aaaatattgt tgatgcgctg gcagtgttcc tgcgccggtt gcattcgatt cctgtttgta
     4141 attgtccttt taacagcgat cgcgtatttc gtctcgctca ggcgcaatca cgaatgaata
     4201 acggtttggt tgatgcgagt gattttgatg acgagcgtaa tggctggcct gttgaacaag
     4261 tctggaaaga aatgcataaa cttttgccat tctcaccgga ttcagtcgtc actcatggtg
     4321 atttctcact tgataacctt atttttgacg aggggaaatt aataggttgt attgatgttg
     4381 gacgagtcgg aatcgcagac cgataccagg atcttgccat cctatggaac tgcctcggtg
     4441 agttttctcc ttcattacag aaacggcttt ttcaaaaata tggtattgat aatcctgata
     4501 tgaataaatt gcagtttcat ttgatgctcg atgagttttt ctaatcagaa ttggttaatt
     4561 ggttgtaacc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     4621 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     4681 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     4741 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt
     4801 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     4861 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     4921 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     4981 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     5041 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     5101 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     5161 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     5221 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     5281 ttttgctcac atgagatctt ggcaggatat attgtggtgt aacgttatca gcttgcatgc
     5341 cggtcgatct agtaacatag atgacaccgc gcgcgataat ttatcctagt ttgcgcgcta
     5401 tattttgttt tctatcgcgt attaaatgta taattgcggg actctaatca taaaaaccca
     5461 tctcataaat aacgtcatgc attacatgtt aattattaca tgcttaacgt aattcaacag
     5521 aaattatatg ataatcatcg caagaccggc aacaggattc aatcttaaga aactttattg
     5581 ccaaatgttt gaacgatctg cttgactcta gctagagtcc gaaccccaga gtcccgctca
     5641 gaagaactcg tcaagaaggc gatagaaggc gatgcgctgc gaatcgggag cggcgatacc
     5701 gtaaagcacg aggaagcggt cagcccattc gccgccaagc tcttcagcaa tatcacgggt
     5761 agccaacgct atgtcctgat agcggtccgc cacacccagc cggccacagt cgatgaatcc
     5821 agaaaagcgg ccattttcca ccatgatatt cggcaagcag gcatcgccgt gggtcacgac
     5881 gagatcctcg ccgtcgggca tccgcgcctt gagcctggcg aacagttcgg ctggcgcgag
     5941 cccctgatgc tcttcgtcca gatcatcctg atcgacaaga ccggcttcca tccgagtacg
     6001 tgctcgctcg atgcgatgtt tcgcttggtg gtcgaatggg caggtagccg gatcaagcgt
     6061 atgcagccgc cgcattgcat cagccatgat ggatactttc tcggcaggag caaggtgaga
     6121 tgacaggaga tcctgccccg gcacttcgcc caatagcagc cagtcccttc ccgcttcagt
     6181 gacaacgtcg agcacagctg cgcaaggaac gcccgtcgtg gccagccacg atagccgcgc
     6241 tgcctcgtct tggagttcat tcagggcacc ggacaggtcg gtcttgacaa aaagaaccgg
     6301 gcgcccctgc gctgacagcc ggaacacggc ggcatcagag cagccgattg tctgttgtgc
     6361 ccagtcatag ccgaatagcc tctccaccca agcggccgga gaacctgcgt gcaatccatc
     6421 ttgttcaatc atgcctcgat cgagttgaga gtgaatatga gactctaatt ggataccgag
     6481 gggaatttat ggaacgtcag tggagcattt ttgacaagaa atatttgcta gctgatagtg
     6541 accttaggcg acttttgaac gcgcaataat ggtttctgac gtatgtgctt agctcattaa
     6601 actccagaaa cccgcggctc agtggctcct tcaacgttgc ggttctgtca gttccaaacg
     6661 taaaacggct tgtcccgcgt catcggcggg ggtcataacg tgactccctt aattctccgc
     6721 tcatgatcga taacattaac gcttacaatt tccattcgcc attcaggctg cgcaactgtt
     6781 gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa gggggatgtg
     6841 ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgt tgtaaaacga
     6901 cggccagtga gcgcgcgtaa tacgactcac tatagggcga attgggtacc gggccccccc
     6961 tcgaggtcga cggtatcgat
//
