LOCUS       sequence-380015-        5497 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Nimble cloning-compatible vector for protein subcellular 
            localization in plants.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5497)
  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 5497)
  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-Green-SubN
FEATURES             Location/Qualifiers
     source          1..5497
                     /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"
     CDS             join(545..547,548..550,551..1261)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(545..547,548..550,551..1261)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(590..611)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(851..870)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1198..1219
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        1443..1545
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             1576..1881
                     /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     1760..1779
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     terminator      1964..2216
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     primer_bind     complement(2234..2250)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2234..2250)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(2287..2307)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2287..2305)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2326..2342)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2326..2342)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2339..2361)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2350..2366
                     /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(2374..2380,2381..2398,2399..2404))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2419..2440
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2557..2574)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2679..2703
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     rep_origin      complement(2794..3382)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2874..2893)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3553..4368)
                     /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             complement(3553..4368)
                     /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     4276..4295
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4360..4379
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4659..5094
                     /label=pSa ori
                     /note="origin of replication from bacterial plasmid pSa"
     misc_feature    5221..5243
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA 
                     (truncated)"
     primer_bind     5393..5415
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5407..5424
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5408..5424
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5434..5453
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5434..5452
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     5477..5493
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     5478..5494
                     /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 tgacatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct
      601 ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac
      661 ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc
      721 caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat
      781 gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat
      841 cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac
      901 cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg
      961 gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa
     1021 gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct
     1081 cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa
     1141 ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat
     1201 ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa
     1261 gcagtggtct ctgtccagtc ctggcctcgt cggccattct cgactaagtt ggcagcatca
     1321 cccgacgcac tttgcgccga ataaatacct gtgacggaag atcacttcgc agaataaata
     1381 aatcctggtg tccctgttga taccgggaag ccctgggcca acttttggcg aaaatgagac
     1441 gttgatcggc acgtaagagg ttccaacttt caccataatg aaataagatc actaccgggc
     1501 gtattttttg agttatcgag attttcagga gctaaggaag ctaaactttt gctgacgaga
     1561 acagggactg gtgaaatgca gtttaaggtt tacacctata aaagagagag ccgttatcgt
     1621 ctgtttgtgg atgtacagag tgatattatt gacacgcctg ggcgacggat ggtgatcccc
     1681 ctggccagtg cacgtctgct gtcagataaa gtctcccgtg aactttaccc ggtggtgcat
     1741 atcggggatg aaagctggcg catgatgacc accgatatgg ccagtgtgcc ggtatccgtt
     1801 atcggggaag aagtggctga tctcagccac cgcgaaaatg acatcaaaaa cgccattaac
     1861 ctgatgttct ggggaatata aatgtcaggc tcccttatac acagggccag tctggccact
     1921 tgtggtctgc tgagaccgtg aggtgaccag ctcgaatttc cccgatcgtt caaacatttg
     1981 gcaataaagt ttcttaagat tgaatcctgt tgccggtctt gcgatgatta tcatataatt
     2041 tctgttgaat tacgttaagc atgtaataat taacatgtaa tgcatgacgt tatttatgag
     2101 atgggttttt atgattagag tcccgcaatt atacatttaa tacgcgatag aaaacaaaat
     2161 atagcgcgca aactaggata aattatcgcg cgcggtgtca tctatgttac tagatcgggc
     2221 ctgcagcccg ggggatccac tagttctaga gcggccgcca ccgcggtgga gctccagctt
     2281 ttgttccctt tagtgagggt taattgcgcg cttggcgtaa tcatggtcat agctgtttcc
     2341 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     2401 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     2461 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     2521 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     2581 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     2641 agaatcaggg gataacgcag gaaagaacat gaaggccttg acaggatata ttggcgggta
     2701 aactaagtcg ctgtatgtgt ttgtttgaga tctcatgtga gcaaaaggcc agcaaaaggc
     2761 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     2821 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     2881 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     2941 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     3001 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     3061 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     3121 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     3181 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     3241 atttggtatc tgcgctctgc tgaagccagt taccttcgga agaagagttg gtagctcttg
     3301 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     3361 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     3421 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     3481 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgtgtaaca
     3541 ttggtctagt gattagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc
     3601 aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc
     3661 gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac
     3721 atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc
     3781 atgagtgacg actgaatccg gtgagaatgg caaaagttta tgcatttctt tccagacttg
     3841 ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt
     3901 cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca
     3961 aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tattttcacc
     4021 tgaatcagga tattcttcta atacctggaa tgctgttttc cctgggatcg cagtggtgag
     4081 taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc
     4141 cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc
     4201 atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcggtaga ttgtcgcacc
     4261 tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga
     4321 atttaatcgc ggccttgagc aagacgtttc ccgttgaata tggctcataa caccccttgt
     4381 attactgttt atgtaagcag acagttttat tgttcatgat gatatatttt tatcttgtgc
     4441 aatgtaacat cagagatttt gagacacaac gtggctttgt tgaataaatc gaacttttgc
     4501 tgagttgaag gatcagatca cgcatcttcc cgacaacgca gaccgttccg tggcaaagca
     4561 aaagttcaaa atcaccaact ggtccaccta caacaaagct ctcatcaacc gtggctccct
     4621 cactttctgg ctggatgatg gggcgattca ggcgatcccc atccaacagc ccgccgtcga
     4681 gcgggctttt ttatccccgg aagcctgtgg atagagggta gttatccacg tgaaaccgct
     4741 aatgccccgc aaagccttga ttcacggggc tttccggccc gctccaaaaa ctatccacgt
     4801 gaaatcgcta atcagggtac gtgaaatcgc taatcggagt acgtgaaatc gctaataagg
     4861 tcacgtgaaa tcgctaatca aaaaggcacg tgagaacgct aatagccctt tcagatcaac
     4921 agcttgcaaa cacccctcgc tccggcaagt agttacagca agtagtatgt tcaattagct
     4981 tttcaattat gaatatatat atcaattatt ggtcgccctt ggcttgtgga caatgcgcta
     5041 cgcgcaccgg ctccgcccgt ggacaaccgc aagcggttgc ccaccgtcga gcgcctttgc
     5101 ccacaacccg gcggccggcc gcaacagatc gttttataaa tttttttttt tgaaaaagaa
     5161 aaagcccgaa aggcggcaac ctctcgggct tctggatttc cgatccccgg aattagatct
     5221 tggcaggata tattgtggtg taacgttaac attaacgctt acaatttcca ttcgccattc
     5281 aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg
     5341 gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca
     5401 cgacgttgta aaacgacggc cagtgagcgc gcgtaatacg actcactata gggcgaattg
     5461 ggtaccgggc cccccctcga ggtcgacggt atcgata
//
