LOCUS       sequence-266221-        5031 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Gateway-compatible entry vector for direct cloning of synthetic 
            trans-acting siRNAs into Arabidopsis thaliana TAS1c precursor at 
            position downstream 3'D1[+].
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5031)
  AUTHORS   Lopez-Dolz L, Spada M, Daros JA, Carbonell A
  TITLE     Fine-tune control of targeted RNAi efficacy by plant artificial 
            small RNAs.
  JOURNAL   Nucleic Acids Res. 2020 May 12. pii: 5836195. doi: 
            10.1093/nar/gkaa343.
  PUBMED    32396204
REFERENCE   2  (bases 1 to 5031)
  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: "Nucleic 
            Acids Res. 2020 May 12. pii: 5836195. doi: 10.1093/nar/gkaa343."
COMMENT     Sequence Label: pENTR-AtTAS1c-D2-B/c
FEATURES             Location/Qualifiers
     source          1..5031
                     /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=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(1111..1116,1117..1134,1135..1141)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             1195..1854
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1261..1280)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             2196..2501
                     /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     2380..2399
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(3156..3255)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(3272..3291)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3273..3291)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3296..3312)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3296..3312)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3414..3433)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3425..4234
                     /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(3492..3511)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      4381..4969
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4870..4889
                     /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 atgagcaatg cttttttata atgccaactt tgtacaaaaa
      661 agcaggctcc gcggccgccc ccttcaccaa acctaaacct aaacggctaa gcccgacgtc
      721 aaataccaaa aagagaaaaa caagagcgcc gtcaagctct gcaaatacga tctgtaagtc
      781 catcttaaca caaaagtgag atgggttctt agatcatgtt ccgccgttag atcgagtcat
      841 ggtcttgtct catagaaagg tactttcgtt tacttctttt gagtatcgag tagagcgtcg
      901 tctatagtta gtttgagatt gcgtttgtca gaagttaggt tcaatgtccc ggtccaattt
      961 tcaccagcca tgtgtcagtt tcgttccttc ccgtcctctt ctttgatttc gttgggttac
     1021 ggatgttttc gagatgaaac agcattgttt tgttgtgatt tttctctaca agcgaataga
     1081 ccatttaaga gaccattagg caccccaggc tttacacttt atgcttccgg ctcgtataat
     1141 gtgtggattt tgagttagga gccgtcgaga ttttcaggag ctaaggaagc taaaatggag
     1201 aaaaaaatca ctggatatac caccgttgat atatcccaat ggcatcgtaa agaacatttt
     1261 gaggcatttc agtcagttgc tcaatgtacc tataaccaga ccgttcagct ggatattacg
     1321 gcctttttaa agaccgtaaa gaaaaataag cacaagtttt atccggcctt tattcacatt
     1381 cttgcccgcc tgatgaatgc tcatccggag ttccgtatgg caatgaaaga cggtgagctg
     1441 gtgatatggg atagtgttca cccttgttac accgttttcc atgagcaaac tgaaacgttt
     1501 tcatcgctct ggagtgaata ccacgacgat ttccggcagt ttctacacat atattcgcaa
     1561 gatgtggcgt gttacggtga aaacctggcc tatttcccta aagggtttat tgagaatatg
     1621 tttttcgtct cagccaatcc ctgggtgagt ttcaccagtt ttgatttaaa cgtggccaat
     1681 atggacaact tcttcgcccc cgttttcacc atgggcaaat attatacgca aggcgacaag
     1741 gtgctgatgc cgctggcgat tcaggttcat catgccgttt gtgatggctt ccatgtcggc
     1801 agaatgctta atgaattaca acagtactgc gatgagtggc agggcggggc gtaaacgcgt
     1861 ggagccggct tactaaaagc cagataacag tatgcgtatt tgcgcgctga tttttgcggt
     1921 ataagaatat atactgatat gtatacccga agtatgtcaa aaagaggtat gctatgaagc
     1981 agcgtattac agtgacagtt gacagcgaca gctatcagtt gctcaaggca tatatgatgt
     2041 caatatctcc ggtctggtaa gcacaaccat gcagaatgaa gcccgtcgtc tgcgtgccga
     2101 acgctggaaa gcggaaaatc aggaagggat ggctgaggtc gcccggttta ttgaaatgaa
     2161 cggctctttt gctgacgaga acaggggctg gtgaaatgca gtttaaggtt tacacctata
     2221 aaagagagag ccgttatcgt ctgtttgtgg atgtacagag tgatattatt gacacgcccg
     2281 gccgacggat ggtgatcccc ctggccagtg cacgtctgct gtcagataaa gtctcccgtg
     2341 aactttaccc ggtggtgcat atcggggatg aaagctggcg catgatgacc accgatatgg
     2401 ccagtgtgcc ggtttccgtt atcggggaag aagtggctga tctcagccac cgcgaaaatg
     2461 acatcaaaaa cgccattaac ctgatgttct ggggaatata aatgtcaggc tcccttatac
     2521 acagccagtc tgcacctcga cggtctcatc ggtggatctt agaaaattat tctaagtcca
     2581 acatagcgta ttctaagttc aacatatcga cgaactagaa aagacattgg acatattcca
     2641 ggatatgcaa aagaaaacaa tgaatattgt tttgaatgtg ttcaagtaaa tgagattttc
     2701 aagtcgtcta aagaacagtt gctaatacag ttacttattt caataaataa ttggttctaa
     2761 taatacaaaa catattcgag gatatgcaga aaaaaagatg tttgttattt tgaaaagctt
     2821 gagtagtttc tctccgaggt gtagcgaaga agcatcatct actttgtaat gtaattttct
     2881 ttatgttttc actttgtaat tttatttgtg ttaatgtacc atggccgata tcggttttat
     2941 tgaaagaaaa tttatgttac ttctgttttg gctttgcaat cagttatgct agttttctta
     3001 taccctttcg taagcttcct aaggaatcgt tcattgattt ccactgcttc attgtatatt
     3061 aaaactttac aactgtatcg accatcatat aattctgggt caagagatga aaatagaaca
     3121 ccacatcgta aagtgaaata agggtgggcg cgccgaccca gctttcttgt acaaagttgg
     3181 cattataaga aagcattgct tatcaatttg ttgcaacgaa caggtcacta tcagtcaaaa
     3241 taaaatcatt atttgccatc cagctgatat cccctatagt gagtcgtatt acatggtcat
     3301 agctgtttcc tggcagctct ggcccgtgtc tcaaaatctc tgatgttaca ttgcacaaga
     3361 taaaaatata tcatcatgaa caataaaact gtctgcttac ataaacagta atacaagggg
     3421 tgttatgagc catattcaac gggaaacgtc gaggccgcga ttaaattcca acatggatgc
     3481 tgatttatat gggtataaat gggctcgcga taatgtcggg caatcaggtg cgacaatcta
     3541 tcgcttgtat gggaagcccg atgcgccaga gttgtttctg aaacatggca aaggtagcgt
     3601 tgccaatgat gttacagatg agatggtcag actaaactgg ctgacggaat ttatgcctct
     3661 tccgaccatc aagcatttta tccgtactcc tgatgatgca tggttactca ccactgcgat
     3721 ccccggaaaa acagcattcc aggtattaga agaatatcct gattcaggtg aaaatattgt
     3781 tgatgcgctg gcagtgttcc tgcgccggtt gcattcgatt cctgtttgta attgtccttt
     3841 taacagcgat cgcgtatttc gtctcgctca ggcgcaatca cgaatgaata acggtttggt
     3901 tgatgcgagt gattttgatg acgagcgtaa tggctggcct gttgaacaag tctggaaaga
     3961 aatgcataaa cttttgccat tctcaccgga ttcagtcgtc actcatggtg atttctcact
     4021 tgataacctt atttttgacg aggggaaatt aataggttgt attgatgttg gacgagtcgg
     4081 aatcgcagac cgataccagg atcttgccat cctatggaac tgcctcggtg agttttctcc
     4141 ttcattacag aaacggcttt ttcaaaaata tggtattgat aatcctgata tgaataaatt
     4201 gcagtttcat ttgatgctcg atgagttttt ctaatcagaa ttggttaatt ggttgtaaca
     4261 ctggcagagc attacgctga cttgacggga cggcgcaagc tcatgaccaa aatcccttaa
     4321 cgtgagttac gcgtcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     4381 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     4441 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     4501 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
     4561 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     4621 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     4681 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     4741 ctacaccgaa ctgagatacc tacagcgtga gcattgagaa agcgccacgc ttcccgaagg
     4801 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     4861 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     4921 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     4981 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt t
//
