LOCUS       sequence-378344-        5621 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 5621)
  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 5621)
  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-Amp-GFP-C
FEATURES             Location/Qualifiers
     source          1..5621
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(24..43)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(25..43)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(50..67)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(50..66)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(59..81)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      207..662
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(294..313)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     504..525
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        740..844
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(845..913,914..1705)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1063..1082)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1876..2464
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2365..2384
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2618..2635
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2752..2773
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2788..2793,2794..2811,2812..2818)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2826..2842
                     /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)."
     primer_bind     2831..2853
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2850..2866
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2850..2866
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        2884..2902
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     2884..2901
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     promoter        3562..3906
                     /label=CaMV 35S promoter
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     primer_bind     3850..3870
                     /label=35S promoter
                     /note="CaMV 35S promoter, forward primer"
     promoter        4175..4277
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             4308..4613
                     /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     4492..4511
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             join(4671..4673,4674..4676,4677..5390)
                     /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(4671..4673,4674..4676,4677..5387)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4716..4737)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4977..4996)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5324..5345
                     /label=EGFP-C
                     /note="EGFP, forward primer"
ORIGIN
        1 catgcgacgt cgggcccaat tcgccctata gtgagtcgta ttacaattca ctggccgtcg
       61 ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac
      121 atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac
      181 agttgcgcag cctgaatggc gaatggacgc gccctgtagc ggcgcattaa gcgcggcggg
      241 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
      301 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
      361 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
      421 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
      481 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
      541 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
      601 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat
      661 ttcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg catcaggtgg
      721 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
      781 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
      841 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
      901 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
      961 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     1021 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     1081 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     1141 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     1201 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     1261 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     1321 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     1381 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     1441 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     1501 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     1561 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     1621 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     1681 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     1741 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     1801 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     1861 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     1921 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     1981 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
     2041 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     2101 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     2161 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     2221 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     2281 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     2341 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     2401 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     2461 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     2521 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     2581 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     2641 ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag
     2701 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag
     2761 ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg
     2821 tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa
     2881 gctatttagg tgacactata gaatactcaa gctatgcatc caacgcgttg ggagctctcc
     2941 catatggtcg actagagcca agctgatctc ctttgccccg gagatcacca tggacgactt
     3001 tctctatctc tacgatctag gaagaaagtt cgacggagaa ggtgacgata ccatgttcac
     3061 caccgataat gagaagatta gcctcttcaa tttcagaaag aatgctgacc cacagatggt
     3121 tagagaggcc tacgcggcag gtctcatcaa gacgatctac ccgagtaata atctccagga
     3181 gatcaaatac cttcccaaga aggttaaaga tgcagtcaaa agattcagga ctaactgcat
     3241 caagaacaca gagaaagata tatttctcaa gatcagaagt actattccag tatggacgat
     3301 tcaaggcttg cttcataaac caaggcaagt aatagagatt ggagtctcta agaaagtagt
     3361 tcctactgaa tcaaaggcca tggagtcaaa aattcagatc gaggatctaa cagaactcgc
     3421 cgtgaagact ggcgaacagt tcatacagag tcttttacga ctcaatgaca agaagaaaat
     3481 cttcgtcaac atggtggagc acgacactct cgtctactcc aagaatatca aagatacagt
     3541 ctcagaagac caaagggcta ttgagacttt tcaacaaagg gtaatatcgg gaaacctcct
     3601 cggattccat tgcccagcta tctgtcactt catcaaaagg acagtagaaa aggaaggtgg
     3661 cacctacaaa tgccatcatt gcgataaagg aaaggctatc gttcaagatg cccctgccga
     3721 cagtggtccc aaagatggac ccccacccac gaggagcatc gtggaaaaag aagacgttcc
     3781 aaccacgtct tcaaagcaag tggattgatg tgatatctcc actgacgtaa gggatgacgc
     3841 acaatcccac tatccttcgc aagacccttc ctctatataa ggaagttcat ttcatttgga
     3901 gaggactccg gtatttttac aacaattacc acaacaaaac aaacaacaaa caacattaca
     3961 atttactatt ctagtcgacc tgcaggcggc cgcaagtggt ctctgtccag tcctggcctc
     4021 gtcggccatt ctcgactaag ttggcagcat cacccgacgc actttgcgcc gaataaatac
     4081 ctgtgacgga agatcacttc gcagaataaa taaatcctgg tgtccctgtt gataccggga
     4141 agccctgggc caacttttgg cgaaaatgag acgttgatcg gcacgtaaga ggttccaact
     4201 ttcaccataa tgaaataaga tcactaccgg gcgtattttt tgagttatcg agattttcag
     4261 gagctaagga agctaaactt ttgctgacga gaacagggac tggtgaaatg cagtttaagg
     4321 tttacaccta taaaagagag agccgttatc gtctgtttgt ggatgtacag agtgatatta
     4381 ttgacacgcc tgggcgacgg atggtgatcc ccctggccag tgcacgtctg ctgtcagata
     4441 aagtctcccg tgaactttac ccggtggtgc atatcgggga tgaaagctgg cgcatgatga
     4501 ccaccgatat ggccagtgtg ccggtatccg ttatcgggga agaagtggct gatctcagcc
     4561 accgcgaaaa tgacatcaaa aacgccatta acctgatgtt ctggggaata taaatgtcag
     4621 gctcccttat acacagggcc agtctggcca cttgtggtct gctgagaccg atggtgagca
     4681 agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa
     4741 acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga
     4801 ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca
     4861 ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact
     4921 tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg
     4981 acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca
     5041 tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt
     5101 acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg
     5161 tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc
     5221 agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca
     5281 cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt
     5341 tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa cggccatgct
     5401 agagtccgca aaaatcacca gtctctctct acaaatctat ctctctctat ttttctccag
     5461 aataatgtgt gagtagttcc cagataaggg aattagggtt cttatagggt ttcgctcatg
     5521 tgttgagcat ataagaaacc cttagtatgt atttgtattt gtaaaatact tctatcaata
     5581 aaatttctaa ttcctaaaac caaaatccag tgacctgcag g
//
