LOCUS       sequence-380044-        6663 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 6663)
  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 6663)
  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-Ecn
FEATURES             Location/Qualifiers
     source          1..6663
                     /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"
     primer_bind     1069..1090
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        1329..1431
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             1462..1767
                     /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     1646..1665
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     complement(2066..2082)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2066..2082)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(2119..2139)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2119..2137)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2158..2174)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2158..2174)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2171..2193)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2182..2198
                     /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(2206..2212,2213..2230,2231..2236))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2251..2272
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2389..2406)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2511..2535
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     rep_origin      2686..3121
                     /label=pSa ori
                     /note="origin of replication from bacterial plasmid pSa"
     primer_bind     complement(3401..3420)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3412..4227
                     /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             3412..4227
                     /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(3485..3504)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      4328..4916
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4817..4836
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    4983..5005
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA 
                     (truncated)"
     terminator      complement(5029..5281)
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     CDS             complement(5321..6115)
                     /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(5432..5451)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     6042..6061
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(6149..6328)
                     /label=NOS promoter
                     /note="nopaline synthase promoter"
     primer_bind     6560..6582
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6575..6591
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6601..6620
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6601..6619
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     6644..6660
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     6645..6661
                     /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 catgggcggc agcgtgcagc tcgccgacca
      961 ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct
     1021 gagctaccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct
     1081 ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca agtccggact
     1141 cagatctcag tggtctctgt ccagtcctgg cctcgtcggc cattctcgac taagttggca
     1201 gcatcacccg acgcactttg cgccgaataa atacctgtga cggaagatca cttcgcagaa
     1261 taaataaatc ctggtgtccc tgttgatacc gggaagccct gggccaactt ttggcgaaaa
     1321 tgagacgttg atcggcacgt aagaggttcc aactttcacc ataatgaaat aagatcacta
     1381 ccgggcgtat tttttgagtt atcgagattt tcaggagcta aggaagctaa acttttgctg
     1441 acgagaacag ggactggtga aatgcagttt aaggtttaca cctataaaag agagagccgt
     1501 tatcgtctgt ttgtggatgt acagagtgat attattgaca cgcctgggcg acggatggtg
     1561 atccccctgg ccagtgcacg tctgctgtca gataaagtct cccgtgaact ttacccggtg
     1621 gtgcatatcg gggatgaaag ctggcgcatg atgaccaccg atatggccag tgtgccggta
     1681 tccgttatcg gggaagaagt ggctgatctc agccaccgcg aaaatgacat caaaaacgcc
     1741 attaacctga tgttctgggg aatataaatg tcaggctccc ttatacacag ggccagtctg
     1801 gccacttgtg gtctgctgag accctgatct agagtccgca aaaatcacca gtctctctct
     1861 acaaatctat ctctctctat ttttctccag aataatgtgt gagtagttcc cagataaggg
     1921 aattagggtt cttatagggt ttcgctcatg tgttgagcat ataagaaacc cttagtatgt
     1981 atttgtattt gtaaaatact tctatcaata aaatttctaa ttcctaaaac caaaatccag
     2041 tgacgcgaat tcctgcagcc cgggggatcc actagttcta gagcggccgc caccgcggtg
     2101 gagctccagc ttttgttccc tttagtgagg gttaattgcg cgcttggcgt aatcatggtc
     2161 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
     2221 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
     2281 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     2341 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
     2401 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     2461 acggttatcc acagaatcag gggataacgc aggaaagaac atgaaggcct tgacaggata
     2521 tattggcggg taaactaagt cgctgtatgt gtttgtttga gatctaattc cggggatcgg
     2581 aaatccagaa gcccgagagg ttgccgcctt tcgggctttt tctttttcaa aaaaaaaaat
     2641 ttataaaacg atctgttgcg gccggccgcc gggttgtggg caaaggcgct cgacggtggg
     2701 caaccgcttg cggttgtcca cgggcggagc cggtgcgcgt agcgcattgt ccacaagcca
     2761 agggcgacca ataattgata tatatattca taattgaaaa gctaattgaa catactactt
     2821 gctgtaacta cttgccggag cgaggggtgt ttgcaagctg ttgatctgaa agggctatta
     2881 gcgttctcac gtgccttttt gattagcgat ttcacgtgac cttattagcg atttcacgta
     2941 ctccgattag cgatttcacg taccctgatt agcgatttca cgtggatagt ttttggagcg
     3001 ggccggaaag ccccgtgaat caaggctttg cggggcatta gcggtttcac gtggataact
     3061 accctctatc cacaggcttc cggggataaa aaagcccgct cgacggcggg ctgttggatg
     3121 gggatcgcct gaatcgcccc atcatccagc cagaaagtga gggagccacg gttgatgaga
     3181 gctttgttgt aggtggacca gttggtgatt ttgaactttt gctttgccac ggaacggtct
     3241 gcgttgtcgg gaagatgcgt gatctgatcc ttcaactcag caaaagttcg atttattcaa
     3301 caaagccacg ttgtggctca aaatctctga tgttacattg cacaagataa aaatatatca
     3361 tcatgaacaa taaaactgtc tgcttacata aacagtaata caaggggtgt tatgagccat
     3421 attcaacggg aaacgtcttg ctcaaggccg cgattaaatt ccaacatgga tgctgattta
     3481 tatgggtata aatgggctcg cgataatgtc gggcaatcag gtgcgacaat ctaccgattg
     3541 tatgggaagc ccgatgcgcc agagttgttt ctgaaacatg gcaaaggtag cgttgccaat
     3601 gatgttacag atgagatggt cagactaaac tggctgacgg aatttatgcc tcttccgacc
     3661 atcaagcatt ttatccgtac tcctgatgat gcatggttac tcaccactgc gatcccaggg
     3721 aaaacagcat tccaggtatt agaagaatat cctgattcag gtgaaaatat tgttgatgcg
     3781 ctggcagtgt tcctgcgccg gttgcattcg attcctgttt gtaattgtcc ttttaacagc
     3841 gatcgcgtat ttcgtctcgc tcaggcgcaa tcacgaatga ataacggttt ggttgatgcg
     3901 agtgattttg atgacgagcg taatggctgg cctgttgaac aagtctggaa agaaatgcat
     3961 aaacttttgc cattctcacc ggattcagtc gtcactcatg gtgatttctc acttgataac
     4021 cttatttttg acgaggggaa attaataggt tgtattgatg ttggacgagt cggaatcgca
     4081 gaccgatacc aggatcttgc catcctatgg aactgcctcg gtgagttttc tccttcatta
     4141 cagaaacggc tttttcaaaa atatggtatt gataatcctg atatgaataa attgcagttt
     4201 catttgatgc tcgatgagtt tttctaatca gaattggtta attggttgta accatgacca
     4261 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     4321 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     4381 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     4441 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc
     4501 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     4561 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     4621 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     4681 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     4741 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     4801 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     4861 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     4921 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgagat
     4981 cttggcagga tatattgtgg tgtaacgtta tcagcttgca tgccggtcga tctagtaaca
     5041 tagatgacac cgcgcgcgat aatttatcct agtttgcgcg ctatattttg ttttctatcg
     5101 cgtattaaat gtataattgc gggactctaa tcataaaaac ccatctcata aataacgtca
     5161 tgcattacat gttaattatt acatgcttaa cgtaattcaa cagaaattat atgataatca
     5221 tcgcaagacc ggcaacagga ttcaatctta agaaacttta ttgccaaatg tttgaacgat
     5281 ctgcttgact ctagctagag tccgaacccc agagtcccgc tcagaagaac tcgtcaagaa
     5341 ggcgatagaa ggcgatgcgc tgcgaatcgg gagcggcgat accgtaaagc acgaggaagc
     5401 ggtcagccca ttcgccgcca agctcttcag caatatcacg ggtagccaac gctatgtcct
     5461 gatagcggtc cgccacaccc agccggccac agtcgatgaa tccagaaaag cggccatttt
     5521 ccaccatgat attcggcaag caggcatcgc cgtgggtcac gacgagatcc tcgccgtcgg
     5581 gcatccgcgc cttgagcctg gcgaacagtt cggctggcgc gagcccctga tgctcttcgt
     5641 ccagatcatc ctgatcgaca agaccggctt ccatccgagt acgtgctcgc tcgatgcgat
     5701 gtttcgcttg gtggtcgaat gggcaggtag ccggatcaag cgtatgcagc cgccgcattg
     5761 catcagccat gatggatact ttctcggcag gagcaaggtg agatgacagg agatcctgcc
     5821 ccggcacttc gcccaatagc agccagtccc ttcccgcttc agtgacaacg tcgagcacag
     5881 ctgcgcaagg aacgcccgtc gtggccagcc acgatagccg cgctgcctcg tcttggagtt
     5941 cattcagggc accggacagg tcggtcttga caaaaagaac cgggcgcccc tgcgctgaca
     6001 gccggaacac ggcggcatca gagcagccga ttgtctgttg tgcccagtca tagccgaata
     6061 gcctctccac ccaagcggcc ggagaacctg cgtgcaatcc atcttgttca atcatgcctc
     6121 gatcgagttg agagtgaata tgagactcta attggatacc gaggggaatt tatggaacgt
     6181 cagtggagca tttttgacaa gaaatatttg ctagctgata gtgaccttag gcgacttttg
     6241 aacgcgcaat aatggtttct gacgtatgtg cttagctcat taaactccag aaacccgcgg
     6301 ctcagtggct ccttcaacgt tgcggttctg tcagttccaa acgtaaaacg gcttgtcccg
     6361 cgtcatcggc gggggtcata acgtgactcc cttaattctc cgctcatgat cgataacatt
     6421 aacgcttaca atttccattc gccattcagg ctgcgcaact gttgggaagg gcgatcggtg
     6481 cgggcctctt cgctattacg ccagctggcg aaagggggat gtgctgcaag gcgattaagt
     6541 tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa cgacggccag tgagcgcgcg
     6601 taatacgact cactataggg cgaattgggt accgggcccc ccctcgaggt cgacggtatc
     6661 gat
//
