LOCUS       sequence-380172-        6653 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 6653)
  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 6653)
  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-Ecc
FEATURES             Location/Qualifiers
     source          1..6653
                     /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"
     primer_bind     1739..1760
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2056..2072)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2056..2072)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(2109..2129)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2109..2127)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2148..2164)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2148..2164)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2161..2183)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2172..2188
                     /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(2196..2202,2203..2220,2221..2226))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2241..2262
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2379..2396)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2501..2525
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     rep_origin      2676..3111
                     /label=pSa ori
                     /note="origin of replication from bacterial plasmid pSa"
     primer_bind     complement(3391..3410)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3402..4217
                     /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             3402..4217
                     /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(3475..3494)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      4318..4906
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4807..4826
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    4973..4995
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA 
                     (truncated)"
     terminator      complement(5019..5271)
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     CDS             complement(5311..6105)
                     /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(5422..5441)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     6032..6051
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(6139..6318)
                     /label=NOS promoter
                     /note="nopaline synthase promoter"
     primer_bind     6550..6572
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6565..6581
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6591..6610
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6591..6609
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     6634..6650
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     6635..6651
                     /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 gagacccggc agcgtgcagc
     1621 tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca
     1681 accactacct gagctaccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca
     1741 tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca
     1801 agtgaagatc cacctgatct agagtccgca aaaatcacca gtctctctct acaaatctat
     1861 ctctctctat ttttctccag aataatgtgt gagtagttcc cagataaggg aattagggtt
     1921 cttatagggt ttcgctcatg tgttgagcat ataagaaacc cttagtatgt atttgtattt
     1981 gtaaaatact tctatcaata aaatttctaa ttcctaaaac caaaatccag tgacgcgaat
     2041 tcctgcagcc cgggggatcc actagttcta gagcggccgc caccgcggtg gagctccagc
     2101 ttttgttccc tttagtgagg gttaattgcg cgcttggcgt aatcatggtc atagctgttt
     2161 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     2221 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     2281 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     2341 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     2401 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     2461 acagaatcag gggataacgc aggaaagaac atgaaggcct tgacaggata tattggcggg
     2521 taaactaagt cgctgtatgt gtttgtttga gatctaattc cggggatcgg aaatccagaa
     2581 gcccgagagg ttgccgcctt tcgggctttt tctttttcaa aaaaaaaaat ttataaaacg
     2641 atctgttgcg gccggccgcc gggttgtggg caaaggcgct cgacggtggg caaccgcttg
     2701 cggttgtcca cgggcggagc cggtgcgcgt agcgcattgt ccacaagcca agggcgacca
     2761 ataattgata tatatattca taattgaaaa gctaattgaa catactactt gctgtaacta
     2821 cttgccggag cgaggggtgt ttgcaagctg ttgatctgaa agggctatta gcgttctcac
     2881 gtgccttttt gattagcgat ttcacgtgac cttattagcg atttcacgta ctccgattag
     2941 cgatttcacg taccctgatt agcgatttca cgtggatagt ttttggagcg ggccggaaag
     3001 ccccgtgaat caaggctttg cggggcatta gcggtttcac gtggataact accctctatc
     3061 cacaggcttc cggggataaa aaagcccgct cgacggcggg ctgttggatg gggatcgcct
     3121 gaatcgcccc atcatccagc cagaaagtga gggagccacg gttgatgaga gctttgttgt
     3181 aggtggacca gttggtgatt ttgaactttt gctttgccac ggaacggtct gcgttgtcgg
     3241 gaagatgcgt gatctgatcc ttcaactcag caaaagttcg atttattcaa caaagccacg
     3301 ttgtggctca aaatctctga tgttacattg cacaagataa aaatatatca tcatgaacaa
     3361 taaaactgtc tgcttacata aacagtaata caaggggtgt tatgagccat attcaacggg
     3421 aaacgtcttg ctcaaggccg cgattaaatt ccaacatgga tgctgattta tatgggtata
     3481 aatgggctcg cgataatgtc gggcaatcag gtgcgacaat ctaccgattg tatgggaagc
     3541 ccgatgcgcc agagttgttt ctgaaacatg gcaaaggtag cgttgccaat gatgttacag
     3601 atgagatggt cagactaaac tggctgacgg aatttatgcc tcttccgacc atcaagcatt
     3661 ttatccgtac tcctgatgat gcatggttac tcaccactgc gatcccaggg aaaacagcat
     3721 tccaggtatt agaagaatat cctgattcag gtgaaaatat tgttgatgcg ctggcagtgt
     3781 tcctgcgccg gttgcattcg attcctgttt gtaattgtcc ttttaacagc gatcgcgtat
     3841 ttcgtctcgc tcaggcgcaa tcacgaatga ataacggttt ggttgatgcg agtgattttg
     3901 atgacgagcg taatggctgg cctgttgaac aagtctggaa agaaatgcat aaacttttgc
     3961 cattctcacc ggattcagtc gtcactcatg gtgatttctc acttgataac cttatttttg
     4021 acgaggggaa attaataggt tgtattgatg ttggacgagt cggaatcgca gaccgatacc
     4081 aggatcttgc catcctatgg aactgcctcg gtgagttttc tccttcatta cagaaacggc
     4141 tttttcaaaa atatggtatt gataatcctg atatgaataa attgcagttt catttgatgc
     4201 tcgatgagtt tttctaatca gaattggtta attggttgta accatgacca aaatccctta
     4261 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     4321 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     4381 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     4441 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
     4501 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     4561 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     4621 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     4681 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     4741 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     4801 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     4861 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     4921 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgagat cttggcagga
     4981 tatattgtgg tgtaacgtta tcagcttgca tgccggtcga tctagtaaca tagatgacac
     5041 cgcgcgcgat aatttatcct agtttgcgcg ctatattttg ttttctatcg cgtattaaat
     5101 gtataattgc gggactctaa tcataaaaac ccatctcata aataacgtca tgcattacat
     5161 gttaattatt acatgcttaa cgtaattcaa cagaaattat atgataatca tcgcaagacc
     5221 ggcaacagga ttcaatctta agaaacttta ttgccaaatg tttgaacgat ctgcttgact
     5281 ctagctagag tccgaacccc agagtcccgc tcagaagaac tcgtcaagaa ggcgatagaa
     5341 ggcgatgcgc tgcgaatcgg gagcggcgat accgtaaagc acgaggaagc ggtcagccca
     5401 ttcgccgcca agctcttcag caatatcacg ggtagccaac gctatgtcct gatagcggtc
     5461 cgccacaccc agccggccac agtcgatgaa tccagaaaag cggccatttt ccaccatgat
     5521 attcggcaag caggcatcgc cgtgggtcac gacgagatcc tcgccgtcgg gcatccgcgc
     5581 cttgagcctg gcgaacagtt cggctggcgc gagcccctga tgctcttcgt ccagatcatc
     5641 ctgatcgaca agaccggctt ccatccgagt acgtgctcgc tcgatgcgat gtttcgcttg
     5701 gtggtcgaat gggcaggtag ccggatcaag cgtatgcagc cgccgcattg catcagccat
     5761 gatggatact ttctcggcag gagcaaggtg agatgacagg agatcctgcc ccggcacttc
     5821 gcccaatagc agccagtccc ttcccgcttc agtgacaacg tcgagcacag ctgcgcaagg
     5881 aacgcccgtc gtggccagcc acgatagccg cgctgcctcg tcttggagtt cattcagggc
     5941 accggacagg tcggtcttga caaaaagaac cgggcgcccc tgcgctgaca gccggaacac
     6001 ggcggcatca gagcagccga ttgtctgttg tgcccagtca tagccgaata gcctctccac
     6061 ccaagcggcc ggagaacctg cgtgcaatcc atcttgttca atcatgcctc gatcgagttg
     6121 agagtgaata tgagactcta attggatacc gaggggaatt tatggaacgt cagtggagca
     6181 tttttgacaa gaaatatttg ctagctgata gtgaccttag gcgacttttg aacgcgcaat
     6241 aatggtttct gacgtatgtg cttagctcat taaactccag aaacccgcgg ctcagtggct
     6301 ccttcaacgt tgcggttctg tcagttccaa acgtaaaacg gcttgtcccg cgtcatcggc
     6361 gggggtcata acgtgactcc cttaattctc cgctcatgat cgataacatt aacgcttaca
     6421 atttccattc gccattcagg ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt
     6481 cgctattacg ccagctggcg aaagggggat gtgctgcaag gcgattaagt tgggtaacgc
     6541 cagggttttc ccagtcacga cgttgtaaaa cgacggccag tgagcgcgcg taatacgact
     6601 cactataggg cgaattgggt accgggcccc ccctcgaggt cgacggtatc gat
//
