LOCUS       sequence-378341-        5622 bp    DNA     circular SYN 15-APR-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 5622)
  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 5622)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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-N
FEATURES             Location/Qualifiers
     source          1..5622
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..48)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(30..48)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(55..72)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(55..71)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(64..86)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      212..667
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(299..318)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     509..530
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        745..849
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(850..918,919..1710)
                     /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(1068..1087)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1881..2469
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2370..2389
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2623..2640
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2757..2778
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2793..2798,2799..2816,2817..2823)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2831..2847
                     /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     2836..2858
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2855..2871
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2855..2871
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        2889..2907
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     2889..2906
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     promoter        3567..3911
                     /label=CaMV 35S promoter
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     primer_bind     3855..3875
                     /label=35S promoter
                     /note="CaMV 35S promoter, forward primer"
     CDS             join(4000..4002,4003..4005,4006..4716)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(4000..4002,4003..4005,4006..4716)
                     /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(4045..4066)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4306..4325)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4653..4674
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        4898..5000
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             5031..5336
                     /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     5215..5234
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
ORIGIN
        1 gcaggcatgc gacgtcgggc ccaattcgcc ctatagtgag tcgtattaca attcactggc
       61 cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc
      121 agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc
      181 ccaacagttg cgcagcctga atggcgaatg gacgcgccct gtagcggcgc attaagcgcg
      241 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
      301 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
      361 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
      421 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
      481 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
      541 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
      601 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt
      661 acaatttcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatca
      721 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
      781 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
      841 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
      901 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
      961 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     1021 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     1081 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     1141 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     1201 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     1261 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     1321 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     1381 accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     1441 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     1501 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     1561 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     1621 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     1681 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     1741 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     1801 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     1861 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     1921 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     1981 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag
     2041 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     2101 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     2161 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     2221 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     2281 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     2341 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     2401 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     2461 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     2521 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     2581 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     2641 gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa
     2701 tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat
     2761 gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg
     2821 ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac
     2881 gccaagctat ttaggtgaca ctatagaata ctcaagctat gcatccaacg cgttgggagc
     2941 tctcccatat ggtcgactag agccaagctg atctcctttg ccccggagat caccatggac
     3001 gactttctct atctctacga tctaggaaga aagttcgacg gagaaggtga cgataccatg
     3061 ttcaccaccg ataatgagaa gattagcctc ttcaatttca gaaagaatgc tgacccacag
     3121 atggttagag aggcctacgc ggcaggtctc atcaagacga tctacccgag taataatctc
     3181 caggagatca aataccttcc caagaaggtt aaagatgcag tcaaaagatt caggactaac
     3241 tgcatcaaga acacagagaa agatatattt ctcaagatca gaagtactat tccagtatgg
     3301 acgattcaag gcttgcttca taaaccaagg caagtaatag agattggagt ctctaagaaa
     3361 gtagttccta ctgaatcaaa ggccatggag tcaaaaattc agatcgagga tctaacagaa
     3421 ctcgccgtga agactggcga acagttcata cagagtcttt tacgactcaa tgacaagaag
     3481 aaaatcttcg tcaacatggt ggagcacgac actctcgtct actccaagaa tatcaaagat
     3541 acagtctcag aagaccaaag ggctattgag acttttcaac aaagggtaat atcgggaaac
     3601 ctcctcggat tccattgccc agctatctgt cacttcatca aaaggacagt agaaaaggaa
     3661 ggtggcacct acaaatgcca tcattgcgat aaaggaaagg ctatcgttca agatgcccct
     3721 gccgacagtg gtcccaaaga tggaccccca cccacgagga gcatcgtgga aaaagaagac
     3781 gttccaacca cgtcttcaaa gcaagtggat tgatgtgata tctccactga cgtaagggat
     3841 gacgcacaat cccactatcc ttcgcaagac ccttcctcta tataaggaag ttcatttcat
     3901 ttggagagga ctccggtatt tttacaacaa ttaccacaac aaaacaaaca acaaacaaca
     3961 ttacaattta ctattctagt cgacctgcag gcggccgcaa tggtgagcaa gggcgaggag
     4021 ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag
     4081 ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc
     4141 atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac
     4201 ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc
     4261 gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac
     4321 aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag
     4381 ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac
     4441 agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag
     4501 atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc
     4561 cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc
     4621 ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc
     4681 gccgggatca ctctcggcat ggacgagctg tacaagcagt ggtctctgtc cagtcctggc
     4741 ctcgtcggcc attctcgact aagttggcag catcacccga cgcactttgc gccgaataaa
     4801 tacctgtgac ggaagatcac ttcgcagaat aaataaatcc tggtgtccct gttgataccg
     4861 ggaagccctg ggccaacttt tggcgaaaat gagacgttga tcggcacgta agaggttcca
     4921 actttcacca taatgaaata agatcactac cgggcgtatt ttttgagtta tcgagatttt
     4981 caggagctaa ggaagctaaa cttttgctga cgagaacagg gactggtgaa atgcagttta
     5041 aggtttacac ctataaaaga gagagccgtt atcgtctgtt tgtggatgta cagagtgata
     5101 ttattgacac gcctgggcga cggatggtga tccccctggc cagtgcacgt ctgctgtcag
     5161 ataaagtctc ccgtgaactt tacccggtgg tgcatatcgg ggatgaaagc tggcgcatga
     5221 tgaccaccga tatggccagt gtgccggtat ccgttatcgg ggaagaagtg gctgatctca
     5281 gccaccgcga aaatgacatc aaaaacgcca ttaacctgat gttctgggga atataaatgt
     5341 caggctccct tatacacagg gccagtctgg ccacttgtgg tctgctgaga ccgtgacggc
     5401 catgctagag tccgcaaaaa tcaccagtct ctctctacaa atctatctct ctctattttt
     5461 ctccagaata atgtgtgagt agttcccaga taagggaatt agggttctta tagggtttcg
     5521 ctcatgtgtt gagcatataa gaaaccctta gtatgtattt gtatttgtaa aatacttcta
     5581 tcaataaaat ttctaattcc taaaaccaaa atccagtgac ct
//
