LOCUS       sequence-454832-        4086 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Entry plasmid with NbmiR482aTS and a ccdB cassette flanked with two 
            inverted BsaI restriction sites. Allows the high throughput cloning 
            of cloning inserts flanked with compatible 4-nt overhangs..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4086)
  AUTHORS   Cisneros AE, Alarcia A, Llorens-Gamez JJ, Puertes A, Juarez-Molina 
            M, Primc A, Carbonell A
  TITLE     Syn-tasiR-VIGS: virus-based targeted RNAi in plants by synthetic 
            trans-acting small interfering RNAs derived from minimal precursors.
  JOURNAL   Nucleic Acids Res. 2025 Feb 27;53(5):gkaf183. doi: 
            10.1093/nar/gkaf183.
  PUBMED    40105245
REFERENCE   2  (bases 1 to 4086)
  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: "Nucleic 
            Acids Res."; date: "2025-02-27"; volume: "53(5):gkaf183. doi"; 
            pages: " 10.1093/nar/gkaf183"
COMMENT     Sequence Label: pENTR-NbmiR482aTS-B/c
FEATURES             Location/Qualifiers
     source          1..4086
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     92..109
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(268..295)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(387..473)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     522..544
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     536..553
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     537..553
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    569..668
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(740..1045)
                     /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     complement(842..861)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(1387..2046)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     1961..1980
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(join(2100..2106,2107..2124,2125..2130))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    complement(2211..2310)
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2327..2346)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2328..2346)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2351..2367)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2351..2367)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2469..2488)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2480..3289
                     /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="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(2547..2566)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3436..4024
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3925..3944
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
       61 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
      121 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
      181 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc
      241 tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta
      301 gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc
      361 acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa
      421 caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg
      481 gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa
      541 aacgacggcc agtcttaagc tcgggcccca aataatgatt ttattttgac tgatagtgac
      601 ctgttcgttg caacaaattg ataagcaatg ctttcttata atgccaactt tgtacaagaa
      661 agctgggtcg gcgcgcccac ccttaatgcc gatgagaccg tcgaggtgca gactggctgt
      721 gtataaggga gcctgacatt tatattcccc agaacatcag gttaatggcg tttttgatgt
      781 cattttcgcg gtggctgaga tcagccactt cttccccgat aacggaaacc ggcacactgg
      841 ccatatcggt ggtcatcatg cgccagcttt catccccgat atgcaccacc gggtaaagtt
      901 cacgggagac tttatctgac agcagacgtg cactggccag ggggatcacc atccgtcggc
      961 cgggcgtgtc aataatatca ctctgtacat ccacaaacag acgataacgg ctctctcttt
     1021 tataggtgta aaccttaaac tgcatttcac cagcccctgt tctcgtcagc aaaagagccg
     1081 ttcatttcaa taaaccgggc gacctcagcc atcccttcct gattttccgc tttccagcgt
     1141 tcggcacgca gacgacgggc ttcattctgc atggttgtgc ttaccagacc ggagatattg
     1201 acatcatata tgccttgagc aactgatagc tgtcgctgtc aactgtcact gtaatacgct
     1261 gcttcatagc atacctcttt ttgacatact tcgggtatac atatcagtat atattcttat
     1321 accgcaaaaa tcagcgcgca aatacgcata ctgttatctg gcttttagta agccggctcc
     1381 acgcgtttac gccccgccct gccactcatc gcagtactgt tgtaattcat taagcattct
     1441 gccgacatgg aagccatcac aaacggcatg atgaacctga atcgccagcg gcatcagcac
     1501 cttgtcgcct tgcgtataat atttgcccat ggtgaaaacg ggggcgaaga agttgtccat
     1561 attggccacg tttaaatcaa aactggtgaa actcacccag ggattggctg agacgaaaaa
     1621 catattctca ataaaccctt tagggaaata ggccaggttt tcaccgtaac acgccacatc
     1681 ttgcgaatat atgtgtagaa actgccggaa atcgtcgtgg tattcactcc agagcgatga
     1741 aaacgtttca gtttgctcat ggaaaacggt gtaacaaggg tgaacactat cccatatcac
     1801 cagctcaccg tctttcattg ccatacggaa ctccggatga gcattcatca ggcgggcaag
     1861 aatgtgaata aaggccggat aaaacttgtg cttatttttc tttacggtct ttaaaaaggc
     1921 cgtaatatcc agctgaacgg tctggttata ggtacattga gcaactgact gaaatgcctc
     1981 aaaatgttct ttacgatgcc attgggatat atcaacggtg gtatatccag tgattttttt
     2041 ctccatttta gcttccttag ctcctgaaaa tctcgacggc tcctaactca aaatccacac
     2101 attatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatggtc tcttaaatgg
     2161 tctattcccg actcccccca taccactaca ggtgaagggg gcggccgcgg agcctgcttt
     2221 tttgtacaaa gttggcatta taaaaaagca ttgctcatca atttgttgca acgaacaggt
     2281 cactatcagt caaaataaaa tcattatttg ccatccagct gatatcccct atagtgagtc
     2341 gtattacatg gtcatagctg tttcctggca gctctggccc gtgtctcaaa atctctgatg
     2401 ttacattgca caagataaaa atatatcatc atgaacaata aaactgtctg cttacataaa
     2461 cagtaataca aggggtgtta tgagccatat tcaacgggaa acgtcgaggc cgcgattaaa
     2521 ttccaacatg gatgctgatt tatatgggta taaatgggct cgcgataatg tcgggcaatc
     2581 aggtgcgaca atctatcgct tgtatgggaa gcccgatgcg ccagagttgt ttctgaaaca
     2641 tggcaaaggt agcgttgcca atgatgttac agatgagatg gtcagactaa actggctgac
     2701 ggaatttatg cctcttccga ccatcaagca ttttatccgt actcctgatg atgcatggtt
     2761 actcaccact gcgatccccg gaaaaacagc attccaggta ttagaagaat atcctgattc
     2821 aggtgaaaat attgttgatg cgctggcagt gttcctgcgc cggttgcatt cgattcctgt
     2881 ttgtaattgt ccttttaaca gcgatcgcgt atttcgtctc gctcaggcgc aatcacgaat
     2941 gaataacggt ttggttgatg cgagtgattt tgatgacgag cgtaatggct ggcctgttga
     3001 acaagtctgg aaagaaatgc ataaactttt gccattctca ccggattcag tcgtcactca
     3061 tggtgatttc tcacttgata accttatttt tgacgagggg aaattaatag gttgtattga
     3121 tgttggacga gtcggaatcg cagaccgata ccaggatctt gccatcctat ggaactgcct
     3181 cggtgagttt tctccttcat tacagaaacg gctttttcaa aaatatggta ttgataatcc
     3241 tgatatgaat aaattgcagt ttcatttgat gctcgatgag tttttctaat cagaattggt
     3301 taattggttg taacactggc agagcattac gctgacttga cgggacggcg caagctcatg
     3361 accaaaatcc cttaacgtga gttacgcgtc gttccactga gcgtcagacc ccgtagaaaa
     3421 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     3481 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     3541 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
     3601 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     3661 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     3721 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     3781 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     3841 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     3901 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     3961 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     4021 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     4081 catgtt
//
