LOCUS       sequence-316662-        3668 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  p3E-Vector (Gateway R2-L3) for Tol2-kit cloning. Vector for 
            performing RNAi (gene silencing - knockdown) in zebrafish via 
            cloning and expression of synthetic miR(s)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3668)
  TITLE     60-p3E_INTRON_EmptyRNAi
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 3668)
  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: 
            "Unpublished"
COMMENT     Sequence Label: 60-p3E_INTRON-EmptyRNAi
FEATURES             Location/Qualifiers
     source          1..3668
                     /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    complement(624..747)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     ncRNA           1061..1087
                     /label=5' miR-155
                     /note="sequence upstream of the precursor of mouse miR-155 
                     microRNA (Uva et al., 2013)"
                     /ncRNA_class="miRNA"
     ncRNA           1119..1160
                     /label=3' miR-155
                     /note="sequence downstream of the precursor of mouse 
                     miR-155 microRNA (Uva et al., 2013)"
                     /ncRNA_class="miRNA"
     polyA_signal    1591..1725
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1615..1634)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1669..1688
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     protein_bind    complement(1777..1872)
                     /gene="mutant version of attL"
                     /label=attL3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1909..1928)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1910..1928)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1933..1949)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1933..1949)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2051..2070)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2062..2871
                     /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(2129..2148)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3018..3606
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3507..3526
                     /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 tcgggccctg cagctctaga gctcgaattc tacaggtcac
      601 taataccatc taagtagttg attcatagtg actgcatatg ttgtgtttta cagtattatg
      661 tagtctgttt tttatgcaaa atctaattta atatattgat atttatatca ttttacgttt
      721 ctcgttcaac tttcttgtac aaagtggctc gaccgatcct gagaacttca gggtgagttt
      781 ggggaccctt gattgttctt tctttttcgc tattgtaaaa ttcatgttat atggaggggg
      841 caaagttttc agggtgttgt ttagaatggg aagatgtccc ttgtatcacc atggaccctc
      901 atgataattt tgtttctttc actttctact ctgttgacaa ccattgtctc ctcttatttt
      961 cttttcattt tctgtaactt tttcgttaaa ctttagcttg catttgtaac gaatttttcg
     1021 tcgatcgttt aaagggaggt agtgagtcga ccagtggatc ctggaggctt gctgaaggct
     1081 gtatgcttga gacggatcgc tagcgatccg tctcacagga cacaaggcct gttactagca
     1141 ctcacatgga acaaatggcc cagatctggc cgcactcgag atatctagac caaattcact
     1201 tttgtttatt tgtcagattg taagtacttt ctctaatcac ttttttttca aggcaatcag
     1261 ggtatattat attgtacttc agcacagttt tagagaacaa ttgttataat taaatgataa
     1321 ggtagaatat ttctgcatat aaattctggc tggcgtggaa atattcttat tggtagaaac
     1381 aactacaccc tggtcatcat cctgcctttc tctttatggt tacaatgata tacactgttt
     1441 gagatgagga taaaatactc tgagtccaaa ccgggcccct ctgctaacca tgttcatgcc
     1501 ttcttctctt tcctacagct cctgggcaac gtgctggttg ttgtgctgtc tcatcatttt
     1561 ggcaaagaat taattcctcg acggatcgta gatccagaca tgataagata cattgatgag
     1621 tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat
     1681 gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc
     1741 attcatttta tgtttcaggt tcagggggag gtgtggcaac tttattatac aaagttggca
     1801 ttataaaaaa gcattgctta tcaatttgtt gcaacgaaca ggtcactatc agtcaaaata
     1861 aaatcattat ttggagctcc atggtagcgt taacgcggcc gcgatatccc ctatagtgag
     1921 tcgtattaca tggtcatagc tgtttcctgg cagctctggc ccgtgtctca aaatctctga
     1981 tgttacattg cacaagataa aaatatatca tcatgaacaa taaaactgtc tgcttacata
     2041 aacagtaata caaggggtgt tatgagccat attcaacggg aaacgtcgag gccgcgatta
     2101 aattccaaca tggatgctga tttatatggg tataaatggg ctcgcgataa tgtcgggcaa
     2161 tcaggtgcga caatctatcg cttgtatggg aagcccgatg cgccagagtt gtttctgaaa
     2221 catggcaaag gtagcgttgc caatgatgtt acagatgaga tggtcagact aaactggctg
     2281 acggaattta tgcctcttcc gaccatcaag cattttatcc gtactcctga tgatgcatgg
     2341 ttactcacca ctgcgatccc cggaaaaaca gcattccagg tattagaaga atatcctgat
     2401 tcaggtgaaa atattgttga tgcgctggca gtgttcctgc gccggttgca ttcgattcct
     2461 gtttgtaatt gtccttttaa cagcgatcgc gtatttcgcc tcgctcaggc gcaatcacga
     2521 atgaataacg gtttggttga tgcgagtgat tttgatgacg agcgtaatgg ctggcctgtt
     2581 gaacaagtct ggaaagaaat gcataaactt ttgccattct caccggattc agtcgtcact
     2641 catggtgatt tctcacttga taaccttatt tttgacgagg ggaaattaat aggttgtatt
     2701 gatgttggac gagtcggaat cgcagaccga taccaggatc ttgccatcct atggaactgc
     2761 ctcggtgagt tttctccttc attacagaaa cggctttttc aaaaatatgg tattgataat
     2821 cctgatatga ataaattgca gtttcatttg atgctcgatg agtttttcta atcagaattg
     2881 gttaattggt tgtaacactg gcagagcatt acgctgactt gacgggacgg cgcaagctca
     2941 tgaccaaaat cccttaacgt gagttacgcg tcgttccact gagcgtcaga ccccgtagaa
     3001 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     3061 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     3121 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg
     3181 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     3241 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     3301 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     3361 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagca ttgagaaagc
     3421 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     3481 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     3541 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     3601 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     3661 cacatgtt
//
