LOCUS       sequence-380017-        5496 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 5496)
  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 5496)
  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-SubC
FEATURES             Location/Qualifiers
     source          1..5496
                     /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"
     promoter        725..827
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             858..1163
                     /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     1042..1061
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             join(1221..1223,1224..1226,1227..1940)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1221..1223,1224..1226,1227..1937)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1266..1287)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1527..1546)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1874..1895
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     terminator      1963..2215
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     primer_bind     complement(2233..2249)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2233..2249)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(2286..2306)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2286..2304)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2325..2341)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2325..2341)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2338..2360)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2349..2365
                     /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(2373..2379,2380..2397,2398..2403))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2418..2439
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2556..2573)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2678..2702
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     rep_origin      complement(2793..3381)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2873..2892)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3552..4367)
                     /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(3552..4367)
                     /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     4275..4294
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4359..4378
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4658..5093
                     /label=pSa ori
                     /note="origin of replication from bacterial plasmid pSa"
     misc_feature    5220..5242
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA 
                     (truncated)"
     primer_bind     5392..5414
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5406..5423
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5407..5423
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5433..5452
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5433..5451
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     5476..5492
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     5477..5493
                     /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 tgacagtggt ctctgtccag tcctggcctc gtcggccatt ctcgactaag ttggcagcat
      601 cacccgacgc actttgcgcc gaataaatac ctgtgacgga agatcacttc gcagaataaa
      661 taaatcctgg tgtccctgtt gataccggga agccctgggc caacttttgg cgaaaatgag
      721 acgttgatcg gcacgtaaga ggttccaact ttcaccataa tgaaataaga tcactaccgg
      781 gcgtattttt tgagttatcg agattttcag gagctaagga agctaaactt ttgctgacga
      841 gaacagggac tggtgaaatg cagtttaagg tttacaccta taaaagagag agccgttatc
      901 gtctgtttgt ggatgtacag agtgatatta ttgacacgcc tgggcgacgg atggtgatcc
      961 ccctggccag tgcacgtctg ctgtcagata aagtctcccg tgaactttac ccggtggtgc
     1021 atatcgggga tgaaagctgg cgcatgatga ccaccgatat ggccagtgtg ccggtatccg
     1081 ttatcgggga agaagtggct gatctcagcc accgcgaaaa tgacatcaaa aacgccatta
     1141 acctgatgtt ctggggaata taaatgtcag gctcccttat acacagggcc agtctggcca
     1201 cttgtggtct gctgagaccg atggtgagca agggcgagga gctgttcacc ggggtggtgc
     1261 ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg
     1321 gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc
     1381 tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc
     1441 gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg
     1501 tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga
     1561 agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg
     1621 acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca
     1681 tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg
     1741 acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg
     1801 tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa gaccccaacg
     1861 agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca
     1921 tggacgagct gtacaagtga ggtgaccagc tcgaatttcc ccgatcgttc aaacatttgg
     1981 caataaagtt tcttaagatt gaatcctgtt gccggtcttg cgatgattat catataattt
     2041 ctgttgaatt acgttaagca tgtaataatt aacatgtaat gcatgacgtt atttatgaga
     2101 tgggttttta tgattagagt cccgcaatta tacatttaat acgcgataga aaacaaaata
     2161 tagcgcgcaa actaggataa attatcgcgc gcggtgtcat ctatgttact agatcgggcc
     2221 tgcagcccgg gggatccact agttctagag cggccgccac cgcggtggag ctccagcttt
     2281 tgttcccttt agtgagggtt aattgcgcgc ttggcgtaat catggtcata gctgtttcct
     2341 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     2401 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     2461 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     2521 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     2581 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     2641 gaatcagggg ataacgcagg aaagaacatg aaggccttga caggatatat tggcgggtaa
     2701 actaagtcgc tgtatgtgtt tgtttgagat ctcatgtgag caaaaggcca gcaaaaggcc
     2761 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     2821 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     2881 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     2941 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     3001 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     3061 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     3121 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     3181 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     3241 tttggtatct gcgctctgct gaagccagtt accttcggaa gaagagttgg tagctcttga
     3301 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     3361 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     3421 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     3481 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgtgtaacat
     3541 tggtctagtg attagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca
     3601 ggattatcaa taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg
     3661 aggcagttcc ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca
     3721 tcaatacaac ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca
     3781 tgagtgacga ctgaatccgg tgagaatggc aaaagtttat gcatttcttt ccagacttgt
     3841 tcaacaggcc agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc
     3901 attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg acaattacaa
     3961 acaggaatcg aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct
     4021 gaatcaggat attcttctaa tacctggaat gctgttttcc ctgggatcgc agtggtgagt
     4081 aaccatgcat catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc
     4141 gtcagccagt ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca
     4201 tgtttcagaa acaactctgg cgcatcgggc ttcccataca atcggtagat tgtcgcacct
     4261 gattgcccga cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa
     4321 tttaatcgcg gccttgagca agacgtttcc cgttgaatat ggctcataac accccttgta
     4381 ttactgttta tgtaagcaga cagttttatt gttcatgatg atatattttt atcttgtgca
     4441 atgtaacatc agagattttg agacacaacg tggctttgtt gaataaatcg aacttttgct
     4501 gagttgaagg atcagatcac gcatcttccc gacaacgcag accgttccgt ggcaaagcaa
     4561 aagttcaaaa tcaccaactg gtccacctac aacaaagctc tcatcaaccg tggctccctc
     4621 actttctggc tggatgatgg ggcgattcag gcgatcccca tccaacagcc cgccgtcgag
     4681 cgggcttttt tatccccgga agcctgtgga tagagggtag ttatccacgt gaaaccgcta
     4741 atgccccgca aagccttgat tcacggggct ttccggcccg ctccaaaaac tatccacgtg
     4801 aaatcgctaa tcagggtacg tgaaatcgct aatcggagta cgtgaaatcg ctaataaggt
     4861 cacgtgaaat cgctaatcaa aaaggcacgt gagaacgcta atagcccttt cagatcaaca
     4921 gcttgcaaac acccctcgct ccggcaagta gttacagcaa gtagtatgtt caattagctt
     4981 ttcaattatg aatatatata tcaattattg gtcgcccttg gcttgtggac aatgcgctac
     5041 gcgcaccggc tccgcccgtg gacaaccgca agcggttgcc caccgtcgag cgcctttgcc
     5101 cacaacccgg cggccggccg caacagatcg ttttataaat tttttttttt gaaaaagaaa
     5161 aagcccgaaa ggcggcaacc tctcgggctt ctggatttcc gatccccgga attagatctt
     5221 ggcaggatat attgtggtgt aacgttaaca ttaacgctta caatttccat tcgccattca
     5281 ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg
     5341 cgaaaggggg atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac
     5401 gacgttgtaa aacgacggcc agtgagcgcg cgtaatacga ctcactatag ggcgaattgg
     5461 gtaccgggcc ccccctcgag gtcgacggta tcgata
//
