LOCUS       sequence-322413-        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  JOURNAL   Unpublished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            "Unpublished"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     3..22
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3..21
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    59..154
                     /gene="mutant version of attL"
                     /label=attL3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     polyA_signal    206..340
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(243..262)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     297..316
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     ncRNA           complement(771..812)
                     /label=3' miR-155
                     /note="sequence downstream of the precursor of mouse 
                     miR-155 microRNA (Uva et al., 2013)"
                     /ncRNA_class="miRNA"
     ncRNA           complement(844..870)
                     /label=5' miR-155
                     /note="sequence upstream of the precursor of mouse miR-155 
                     microRNA (Uva et al., 2013)"
                     /ncRNA_class="miRNA"
     protein_bind    1184..1307
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1378..1395)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1378..1394)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1387..1409)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     terminator      1458..1544
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1636..1663
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     complement(1822..1839)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1993..2581)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2073..2092)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2728..3537)
                     /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     3451..3470
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3529..3548
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     3650..3666
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3650..3666
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 tgtaatacga ctcactatag gggatatcgc ggccgcgtta acgctaccat ggagctccaa
       61 ataatgattt tattttgact gatagtgacc tgttcgttgc aacaaattga taagcaatgc
      121 ttttttataa tgccaacttt gtataataaa gttgccacac ctccccctga acctgaaaca
      181 taaaatgaat gcaattgttg ttgttaactt gtttattgca gcttataatg gttacaaata
      241 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
      301 tttgtccaaa ctcatcaatg tatcttatca tgtctggatc tacgatccgt cgaggaatta
      361 attctttgcc aaaatgatga gacagcacaa caaccagcac gttgcccagg agctgtagga
      421 aagagaagaa ggcatgaaca tggttagcag aggggcccgg tttggactca gagtatttta
      481 tcctcatctc aaacagtgta tatcattgta accataaaga gaaaggcagg atgatgacca
      541 gggtgtagtt gtttctacca ataagaatat ttccacgcca gccagaattt atatgcagaa
      601 atattctacc ttatcattta attataacaa ttgttctcta aaactgtgct gaagtacaat
      661 ataatatacc ctgattgcct tgaaaaaaaa gtgattagag aaagtactta caatctgaca
      721 aataaacaaa agtgaatttg gtctagatat ctcgagtgcg gccagatctg ggccatttgt
      781 tccatgtgag tgctagtaac aggccttgtg tcctgtgaga cggatcgcta gcgatccgtc
      841 tcaagcatac agccttcagc aagcctccag gatccactgg tcgactcact acctcccttt
      901 aaacgatcga cgaaaaattc gttacaaatg caagctaaag tttaacgaaa aagttacaga
      961 aaatgaaaag aaaataagag gagacaatgg ttgtcaacag agtagaaagt gaaagaaaca
     1021 aaattatcat gagggtccat ggtgatacaa gggacatctt cccattctaa acaacaccct
     1081 gaaaactttg ccccctccat ataacatgaa ttttacaata gcgaaaaaga aagaacaatc
     1141 aagggtcccc aaactcaccc tgaagttctc aggatcggtc gagccacttt gtacaagaaa
     1201 gttgaacgag aaacgtaaaa tgatataaat atcaatatat taaattagat tttgcataaa
     1261 aaacagacta cataatactg taaaacacaa catatgcagt cactatgaac caactactta
     1321 gatggtatta gtgacctgta gaattcgagc tctagagctg cagggcccga gcttaagact
     1381 ggccgtcgtt ttacaacgtc gtgactggga aaacatccat gctagcgtta acgcgagagt
     1441 agggaactgc caggcatcaa ataaaacgaa aggctcagtc ggaagactgg gcctttcgtt
     1501 ttatctgttg tttgtcggtg aacgctctcc tgagtaggac aaatccgccg ggagcggatt
     1561 tgaacgttgt gaagcaacgg cccggagggt ggcgggcagg acgcccgcca taaactgcca
     1621 ggcatcaaac taagcagaag gccatcctga cggatggcct ttttgcgttt ctacaaactc
     1681 ttcctggcta gcggtacgcg tattaattgc gttgcgctca ctgcccgctt tccagtcggg
     1741 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     1801 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     1861 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     1921 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     1981 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     2041 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     2101 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     2161 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     2221 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     2281 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     2341 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     2401 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     2461 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     2521 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     2581 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgacg cgtaactcac
     2641 gttaagggat tttggtcatg agcttgcgcc gtcccgtcaa gtcagcgtaa tgctctgcca
     2701 gtgttacaac caattaacca attctgatta gaaaaactca tcgagcatca aatgaaactg
     2761 caatttattc atatcaggat tatcaatacc atatttttga aaaagccgtt tctgtaatga
     2821 aggagaaaac tcaccgaggc agttccatag gatggcaaga tcctggtatc ggtctgcgat
     2881 tccgactcgt ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa taaggttatc
     2941 aagtgagaaa tcaccatgag tgacgactga atccggtgag aatggcaaaa gtttatgcat
     3001 ttctttccag acttgttcaa caggccagcc attacgctcg tcatcaaaat cactcgcatc
     3061 aaccaaaccg ttattcattc gtgattgcgc ctgagcgagg cgaaatacgc gatcgctgtt
     3121 aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc aggaacactg ccagcgcatc
     3181 aacaatattt tcacctgaat caggatattc ttctaatacc tggaatgctg tttttccggg
     3241 gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg ataaaatgct tgatggtcgg
     3301 aagaggcata aattccgtca gccagtttag tctgaccatc tcatctgtaa catcattggc
     3361 aacgctacct ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc catacaagcg
     3421 atagattgtc gcacctgatt gcccgacatt atcgcgagcc catttatacc catataaatc
     3481 agcatccatg ttggaattta atcgcggcct cgacgtttcc cgttgaatat ggctcataac
     3541 accccttgta ttactgttta tgtaagcaga cagttttatt gttcatgatg atatattttt
     3601 atcttgtgca atgtaacatc agagattttg agacacgggc cagagctgcc aggaaacagc
     3661 tatgacca
//
