LOCUS       sequence-454806-        4049 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Entry plasmid with 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 4049)
  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 4049)
  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-B/c
FEATURES             Location/Qualifiers
     source          1..4049
                     /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(736..1041)
                     /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(838..857)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(1383..2042)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     1957..1976
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(join(2096..2102,2103..2120,2121..2126))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    complement(2174..2273)
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2290..2309)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2291..2309)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2314..2330)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2314..2330)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2432..2451)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2443..3252
                     /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(2510..2529)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3399..3987
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3888..3907
                     /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 ccttaatgtg agaccgtcga ggtgcagact ggctgtgtat
      721 aagggagcct gacatttata ttccccagaa catcaggtta atggcgtttt tgatgtcatt
      781 ttcgcggtgg ctgagatcag ccacttcttc cccgataacg gaaaccggca cactggccat
      841 atcggtggtc atcatgcgcc agctttcatc cccgatatgc accaccgggt aaagttcacg
      901 ggagacttta tctgacagca gacgtgcact ggccaggggg atcaccatcc gtcggccggg
      961 cgtgtcaata atatcactct gtacatccac aaacagacga taacggctct ctcttttata
     1021 ggtgtaaacc ttaaactgca tttcaccagc ccctgttctc gtcagcaaaa gagccgttca
     1081 tttcaataaa ccgggcgacc tcagccatcc cttcctgatt ttccgctttc cagcgttcgg
     1141 cacgcagacg acgggcttca ttctgcatgg ttgtgcttac cagaccggag atattgacat
     1201 catatatgcc ttgagcaact gatagctgtc gctgtcaact gtcactgtaa tacgctgctt
     1261 catagcatac ctctttttga catacttcgg gtatacatat cagtatatat tcttataccg
     1321 caaaaatcag cgcgcaaata cgcatactgt tatctggctt ttagtaagcc ggctccacgc
     1381 gtttacgccc cgccctgcca ctcatcgcag tactgttgta attcattaag cattctgccg
     1441 acatggaagc catcacaaac ggcatgatga acctgaatcg ccagcggcat cagcaccttg
     1501 tcgccttgcg tataatattt gcccatggtg aaaacggggg cgaagaagtt gtccatattg
     1561 gccacgttta aatcaaaact ggtgaaactc acccagggat tggctgagac gaaaaacata
     1621 ttctcaataa accctttagg gaaataggcc aggttttcac cgtaacacgc cacatcttgc
     1681 gaatatatgt gtagaaactg ccggaaatcg tcgtggtatt cactccagag cgatgaaaac
     1741 gtttcagttt gctcatggaa aacggtgtaa caagggtgaa cactatccca tatcaccagc
     1801 tcaccgtctt tcattgccat acggaactcc ggatgagcat tcatcaggcg ggcaagaatg
     1861 tgaataaagg ccggataaaa cttgtgctta tttttcttta cggtctttaa aaaggccgta
     1921 atatccagct gaacggtctg gttataggta cattgagcaa ctgactgaaa tgcctcaaaa
     1981 tgttctttac gatgccattg ggatatatca acggtggtat atccagtgat ttttttctcc
     2041 attttagctt ccttagctcc tgaaaatctc gacggctcct aactcaaaat ccacacatta
     2101 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atggtctctt acaggtgaag
     2161 ggggcggccg cggagcctgc ttttttgtac aaagttggca ttataaaaaa gcattgctca
     2221 tcaatttgtt gcaacgaaca ggtcactatc agtcaaaata aaatcattat ttgccatcca
     2281 gctgatatcc cctatagtga gtcgtattac atggtcatag ctgtttcctg gcagctctgg
     2341 cccgtgtctc aaaatctctg atgttacatt gcacaagata aaaatatatc atcatgaaca
     2401 ataaaactgt ctgcttacat aaacagtaat acaaggggtg ttatgagcca tattcaacgg
     2461 gaaacgtcga ggccgcgatt aaattccaac atggatgctg atttatatgg gtataaatgg
     2521 gctcgcgata atgtcgggca atcaggtgcg acaatctatc gcttgtatgg gaagcccgat
     2581 gcgccagagt tgtttctgaa acatggcaaa ggtagcgttg ccaatgatgt tacagatgag
     2641 atggtcagac taaactggct gacggaattt atgcctcttc cgaccatcaa gcattttatc
     2701 cgtactcctg atgatgcatg gttactcacc actgcgatcc ccggaaaaac agcattccag
     2761 gtattagaag aatatcctga ttcaggtgaa aatattgttg atgcgctggc agtgttcctg
     2821 cgccggttgc attcgattcc tgtttgtaat tgtcctttta acagcgatcg cgtatttcgt
     2881 ctcgctcagg cgcaatcacg aatgaataac ggtttggttg atgcgagtga ttttgatgac
     2941 gagcgtaatg gctggcctgt tgaacaagtc tggaaagaaa tgcataaact tttgccattc
     3001 tcaccggatt cagtcgtcac tcatggtgat ttctcacttg ataaccttat ttttgacgag
     3061 gggaaattaa taggttgtat tgatgttgga cgagtcggaa tcgcagaccg ataccaggat
     3121 cttgccatcc tatggaactg cctcggtgag ttttctcctt cattacagaa acggcttttt
     3181 caaaaatatg gtattgataa tcctgatatg aataaattgc agtttcattt gatgctcgat
     3241 gagtttttct aatcagaatt ggttaattgg ttgtaacact ggcagagcat tacgctgact
     3301 tgacgggacg gcgcaagctc atgaccaaaa tcccttaacg tgagttacgc gtcgttccac
     3361 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     3421 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     3481 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     3541 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     3601 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     3661 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     3721 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     3781 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     3841 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     3901 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     3961 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     4021 gccttttgct ggccttttgc tcacatgtt
//
