LOCUS       sequence-379582-        6984 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 6984)
  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 6984)
  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-Enn
FEATURES             Location/Qualifiers
     source          1..6984
                     /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"
     CDS             join(932..934,935..937,938..1450)
                     /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(977..998)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1238..1257)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     promoter        1650..1752
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             1783..2088
                     /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     1967..1986
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     complement(2387..2403)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2387..2403)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(2440..2460)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2440..2458)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2479..2495)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2479..2495)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2492..2514)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2503..2519
                     /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(2527..2533,2534..2551,2552..2557))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2572..2593
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2710..2727)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2832..2856
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     rep_origin      3007..3442
                     /label=pSa ori
                     /note="origin of replication from bacterial plasmid pSa"
     primer_bind     complement(3722..3741)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3733..4548
                     /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             3733..4548
                     /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(3806..3825)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      4649..5237
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5138..5157
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    5304..5326
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA 
                     (truncated)"
     terminator      complement(5350..5602)
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     CDS             complement(5642..6436)
                     /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(5753..5772)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     6363..6382
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(6470..6649)
                     /label=NOS promoter
                     /note="nopaline synthase promoter"
     primer_bind     6881..6903
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6896..6912
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6922..6941
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6922..6940
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     6965..6981
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     6966..6982
                     /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 catggtgagc aagggcgagg agctgttcac
      961 cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt
     1021 gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac
     1081 caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accttcggct acggcctgca
     1141 gtgcttcgcc cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc
     1201 cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg
     1261 cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga
     1321 cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa
     1381 cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca
     1441 caacatcgag gactccggac tcagatctca gtggtctctg tccagtcctg gcctcgtcgg
     1501 ccattctcga ctaagttggc agcatcaccc gacgcacttt gcgccgaata aatacctgtg
     1561 acggaagatc acttcgcaga ataaataaat cctggtgtcc ctgttgatac cgggaagccc
     1621 tgggccaact tttggcgaaa atgagacgtt gatcggcacg taagaggttc caactttcac
     1681 cataatgaaa taagatcact accgggcgta ttttttgagt tatcgagatt ttcaggagct
     1741 aaggaagcta aacttttgct gacgagaaca gggactggtg aaatgcagtt taaggtttac
     1801 acctataaaa gagagagccg ttatcgtctg tttgtggatg tacagagtga tattattgac
     1861 acgcctgggc gacggatggt gatccccctg gccagtgcac gtctgctgtc agataaagtc
     1921 tcccgtgaac tttacccggt ggtgcatatc ggggatgaaa gctggcgcat gatgaccacc
     1981 gatatggcca gtgtgccggt atccgttatc ggggaagaag tggctgatct cagccaccgc
     2041 gaaaatgaca tcaaaaacgc cattaacctg atgttctggg gaatataaat gtcaggctcc
     2101 cttatacaca gggccagtct ggccacttgt ggtctgctga gaccctgatc tagagtccgc
     2161 aaaaatcacc agtctctctc tacaaatcta tctctctcta tttttctcca gaataatgtg
     2221 tgagtagttc ccagataagg gaattagggt tcttataggg tttcgctcat gtgttgagca
     2281 tataagaaac ccttagtatg tatttgtatt tgtaaaatac ttctatcaat aaaatttcta
     2341 attcctaaaa ccaaaatcca gtgacgcgaa ttcctgcagc ccgggggatc cactagttct
     2401 agagcggccg ccaccgcggt ggagctccag cttttgttcc ctttagtgag ggttaattgc
     2461 gcgcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     2521 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     2581 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     2641 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     2701 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     2761 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     2821 catgaaggcc ttgacaggat atattggcgg gtaaactaag tcgctgtatg tgtttgtttg
     2881 agatctaatt ccggggatcg gaaatccaga agcccgagag gttgccgcct ttcgggcttt
     2941 ttctttttca aaaaaaaaaa tttataaaac gatctgttgc ggccggccgc cgggttgtgg
     3001 gcaaaggcgc tcgacggtgg gcaaccgctt gcggttgtcc acgggcggag ccggtgcgcg
     3061 tagcgcattg tccacaagcc aagggcgacc aataattgat atatatattc ataattgaaa
     3121 agctaattga acatactact tgctgtaact acttgccgga gcgaggggtg tttgcaagct
     3181 gttgatctga aagggctatt agcgttctca cgtgcctttt tgattagcga tttcacgtga
     3241 ccttattagc gatttcacgt actccgatta gcgatttcac gtaccctgat tagcgatttc
     3301 acgtggatag tttttggagc gggccggaaa gccccgtgaa tcaaggcttt gcggggcatt
     3361 agcggtttca cgtggataac taccctctat ccacaggctt ccggggataa aaaagcccgc
     3421 tcgacggcgg gctgttggat ggggatcgcc tgaatcgccc catcatccag ccagaaagtg
     3481 agggagccac ggttgatgag agctttgttg taggtggacc agttggtgat tttgaacttt
     3541 tgctttgcca cggaacggtc tgcgttgtcg ggaagatgcg tgatctgatc cttcaactca
     3601 gcaaaagttc gatttattca acaaagccac gttgtggctc aaaatctctg atgttacatt
     3661 gcacaagata aaaatatatc atcatgaaca ataaaactgt ctgcttacat aaacagtaat
     3721 acaaggggtg ttatgagcca tattcaacgg gaaacgtctt gctcaaggcc gcgattaaat
     3781 tccaacatgg atgctgattt atatgggtat aaatgggctc gcgataatgt cgggcaatca
     3841 ggtgcgacaa tctaccgatt gtatgggaag cccgatgcgc cagagttgtt tctgaaacat
     3901 ggcaaaggta gcgttgccaa tgatgttaca gatgagatgg tcagactaaa ctggctgacg
     3961 gaatttatgc ctcttccgac catcaagcat tttatccgta ctcctgatga tgcatggtta
     4021 ctcaccactg cgatcccagg gaaaacagca ttccaggtat tagaagaata tcctgattca
     4081 ggtgaaaata ttgttgatgc gctggcagtg ttcctgcgcc ggttgcattc gattcctgtt
     4141 tgtaattgtc cttttaacag cgatcgcgta tttcgtctcg ctcaggcgca atcacgaatg
     4201 aataacggtt tggttgatgc gagtgatttt gatgacgagc gtaatggctg gcctgttgaa
     4261 caagtctgga aagaaatgca taaacttttg ccattctcac cggattcagt cgtcactcat
     4321 ggtgatttct cacttgataa ccttattttt gacgagggga aattaatagg ttgtattgat
     4381 gttggacgag tcggaatcgc agaccgatac caggatcttg ccatcctatg gaactgcctc
     4441 ggtgagtttt ctccttcatt acagaaacgg ctttttcaaa aatatggtat tgataatcct
     4501 gatatgaata aattgcagtt tcatttgatg ctcgatgagt ttttctaatc agaattggtt
     4561 aattggttgt aaccatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     4621 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     4681 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     4741 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
     4801 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     4861 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     4921 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     4981 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     5041 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     5101 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     5161 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     5221 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     5281 ggccttttgc tcacatgaga tcttggcagg atatattgtg gtgtaacgtt atcagcttgc
     5341 atgccggtcg atctagtaac atagatgaca ccgcgcgcga taatttatcc tagtttgcgc
     5401 gctatatttt gttttctatc gcgtattaaa tgtataattg cgggactcta atcataaaaa
     5461 cccatctcat aaataacgtc atgcattaca tgttaattat tacatgctta acgtaattca
     5521 acagaaatta tatgataatc atcgcaagac cggcaacagg attcaatctt aagaaacttt
     5581 attgccaaat gtttgaacga tctgcttgac tctagctaga gtccgaaccc cagagtcccg
     5641 ctcagaagaa ctcgtcaaga aggcgataga aggcgatgcg ctgcgaatcg ggagcggcga
     5701 taccgtaaag cacgaggaag cggtcagccc attcgccgcc aagctcttca gcaatatcac
     5761 gggtagccaa cgctatgtcc tgatagcggt ccgccacacc cagccggcca cagtcgatga
     5821 atccagaaaa gcggccattt tccaccatga tattcggcaa gcaggcatcg ccgtgggtca
     5881 cgacgagatc ctcgccgtcg ggcatccgcg ccttgagcct ggcgaacagt tcggctggcg
     5941 cgagcccctg atgctcttcg tccagatcat cctgatcgac aagaccggct tccatccgag
     6001 tacgtgctcg ctcgatgcga tgtttcgctt ggtggtcgaa tgggcaggta gccggatcaa
     6061 gcgtatgcag ccgccgcatt gcatcagcca tgatggatac tttctcggca ggagcaaggt
     6121 gagatgacag gagatcctgc cccggcactt cgcccaatag cagccagtcc cttcccgctt
     6181 cagtgacaac gtcgagcaca gctgcgcaag gaacgcccgt cgtggccagc cacgatagcc
     6241 gcgctgcctc gtcttggagt tcattcaggg caccggacag gtcggtcttg acaaaaagaa
     6301 ccgggcgccc ctgcgctgac agccggaaca cggcggcatc agagcagccg attgtctgtt
     6361 gtgcccagtc atagccgaat agcctctcca cccaagcggc cggagaacct gcgtgcaatc
     6421 catcttgttc aatcatgcct cgatcgagtt gagagtgaat atgagactct aattggatac
     6481 cgaggggaat ttatggaacg tcagtggagc atttttgaca agaaatattt gctagctgat
     6541 agtgacctta ggcgactttt gaacgcgcaa taatggtttc tgacgtatgt gcttagctca
     6601 ttaaactcca gaaacccgcg gctcagtggc tccttcaacg ttgcggttct gtcagttcca
     6661 aacgtaaaac ggcttgtccc gcgtcatcgg cgggggtcat aacgtgactc ccttaattct
     6721 ccgctcatga tcgataacat taacgcttac aatttccatt cgccattcag gctgcgcaac
     6781 tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga
     6841 tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa
     6901 acgacggcca gtgagcgcgc gtaatacgac tcactatagg gcgaattggg taccgggccc
     6961 cccctcgagg tcgacggtat cgat
//
