LOCUS       sequence-71778-e        4058 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  To create an AttR3-AttL4 flanked Entry vector encoding a 
            miR30-embedded shRNA to knockdown targeted gene expression. (This 
            corresponds to a module 4 plasmid)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 4058)
  AUTHORS   Geiling B, Vandal G, Posner AR, de Bruyns A, Dutchak KL, Garnett S, 
            Dankort D
  TITLE     A modular lentiviral and retroviral construction system to rapidly 
            generate vectors for gene expression and gene knockdown in vitro and
            in vivo.
  JOURNAL   PLoS One. 2013 Oct 11;8(10):e76279. doi: 
            10.1371/journal.pone.0076279.
  PUBMED    24146852
REFERENCE   2  (bases 1 to 4058)
  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: "PLoS One.";
            date: "2013-10-11"; volume: "8(10):e76279. doi"; pages: " 
            10.1371/journal.pone.0076279"
COMMENT     Sequence Label: pBEG R3-miRNA(ccdB)-L4   #BGV228
FEATURES             Location/Qualifiers
     source          1..4058
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     protein_bind    102..123
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     protein_bind    178..194
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     183..205
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     202..218
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     202..218
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    complement(288..411)
                     /gene="mutant version of attR"
                     /label=attR3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     ncRNA           457..550
                     /label=5' miR-30a
                     /note="sequence upstream of the 71-nt precursor of the 
                     human miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     promoter        join(571..576,577..594,595..601)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             655..1314
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(721..740)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             1656..1961
                     /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
                     VPRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     1840..1859
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     ncRNA           2005..2117
                     /label=3' miR-30a
                     /note="sequence downstream of the 71-nt precursor of the 
                     human miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     protein_bind    complement(2176..2271)
                     /gene="mutant version of attL"
                     /label=attL4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2300..2319)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2301..2319)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2326..2343)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2326..2342)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2335..2357)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             complement(2549..3367)
                     /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="MSHIQRETSCSSRPRLNSNMDADLYGYKWARDNVGQSGATIY*LY
                     GKPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPG
                     KTAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVD
                     ASDFDDERNGWPVEQVWKEMHNLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVG
                     IADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     3272..3291
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3359..3378
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3484..4028
                     /direction=RIGHT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     3929..3948
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac
       61 aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact
      121 cattaggcac cccaggcttt acactttatg cttccgcggc tcgtatgttg tgtggaattg
      181 tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc actagcagat
      241 ctgctagact ttccctcagc ggatctgcta gtgcggccgc gccggcccat agtgactgga
      301 tatgttgtgt tttacagtat tatgtagtct gttttttatg caaaatctaa tttaatatat
      361 tgatatttat atcattttac gtttctcgtt caactttatt atacatagtt ggctagtgga
      421 tccagcgctg gcgcgccatt taaatcgata gtttgtttga atgaggcttc agtactttac
      481 agaatcgttg cctgcacatc ttggaaacac ttgctgggat tacttcttca ggttaaccca
      541 acagaaggct cgagattagg caccccaggc tttacacttt atgcttccgg ctcgtataat
      601 gtgtggattt tgagttagga tccgtcgaga ttttcaggag ctaaggaagc taaaatggag
      661 aaaaaaatca ctggatatac caccgttgat atatcccaat ggcatcgtaa agaacatttt
      721 gaggcatttc agtcagttgc tcaatgtacc tataaccaga ccgttcagct ggatattacg
      781 gcctttttaa agaccgtaaa gaaaaataag cacaagtttt atccggcctt tattcacatt
      841 cttgcccgcc tgatgaatgc tcatccggag ttccgtatgg caatgaaaga cggtgagctg
      901 gtgatatggg atagtgttca cccttgttac accgttttcc atgagcaaac tgaaacgttt
      961 tcatcgctct ggagtgaata ccacgacgat ttccggcagt ttctacacat atattcgcaa
     1021 gatgtggcgt gttacggtga aaacctggcc tatttcccta aagggtttat tgagaatatg
     1081 tttttcgtct cagccaatcc ctgggtgagt ttcaccagtt ttgatttaaa cgtggccaat
     1141 atggacaact tcttcgcccc cgttttcacc atgggcaaat attatacgca aggcgacaag
     1201 gtgctgatgc cgctggcgat tcaggttcat catgccgtct gtgatggctt ccatgtcggc
     1261 agaatgctta atgaattaca acagtactgc gatgagtggc agggcggggc gtaatctaga
     1321 ggatccggct tactaaaagc cagataacag tatgcgtatt tgcgcgctga tttttgcggt
     1381 ataagaatat atactgatat gtatacccga agtatgtcaa aaagaggtgt gctatgaagc
     1441 agcgtattac agtgacagtt gacagcgaca gctatcagtt gctcaaggca tatatgatgt
     1501 caatatctcc ggtctggtaa gcacaaccat gcagaatgaa gcccgtcgtc tgcgtgccga
     1561 acgctggaaa gcggaaaatc aggaagggat ggctgaggtc gcccggttta ttgaaatgaa
     1621 cggctctttt gctgacgaga acagggactg gtgaaatgca gtttaaggtt tacacctata
     1681 aaagagagag ccgttatcgt ctgtttgtgg atgtacagag tgatattatt gacacgcccg
     1741 ggcgacggat ggtgatcccc ctggccagtg cacgtctgct gtcagataaa gtcccccgtg
     1801 aactttaccc ggtggtgcat atcggggatg aaagctggcg catgatgacc accgatatgg
     1861 ccagtgtgcc ggtctccgtt atcggggaag aagtggctga tctcagccac cgcgaaaatg
     1921 acatcaaaaa cgccattaac ctgatgttct ggggaatata aatgtcaggc tcccttatac
     1981 acagccagtc tgcaggtcga cgaattcaag gggctacttt aggagcaatt atcttgttta
     2041 ctaaaactga ataccttgct atctctttga tacattttta caaagctgaa ttaaaatggt
     2101 ataaattaaa tcactttttt caattggttt aaaccctgca ggtacgtaac gcgtggatat
     2161 cggcgcgccc ccgggcaact tttctataca aagttggcat tataagaaag cattgcttat
     2221 caatttgttg caacgaacag gtcactatca gtcaaaataa aatcattatt tactagtgtt
     2281 aacttcgatc cccaattcgc cctatagtga gtcgtattac aattcactgg ccgtcgtttt
     2341 acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc
     2401 ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt
     2461 gcgcagcctg aatggcgaat gagcttgcgc cgtcccgtca agtcagcgta atgctctgcc
     2521 agtgttacaa ccaattaacc aattctgatt agaaaaactc atcgagcatc aaatgaaact
     2581 gcaatttatt catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg
     2641 aaggagaaaa ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga
     2701 ttccgactcg tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat
     2761 caagtgagaa atcaccatga gtgacgactg aatccggtga gaatggcaaa aggttatgca
     2821 tttctttcca gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat
     2881 caaccaaacc gttattcatt cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt
     2941 taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat
     3001 caacaatatt ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccag
     3061 ggatcgcagt ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg
     3121 gaagaggcat aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg
     3181 caacgctacc tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc
     3241 aatagattgt cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat
     3301 cagcatccat gttggaattt aatcgcggcc tcgacgagca agacgtttcc cgttgaatat
     3361 ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt gttcatgatg
     3421 atatattttt atcttgtgca atgtaacatc agagattttg agacactcga caagatgatc
     3481 ttcttgagat cgttttggtc tgcgcgtaat ctcttgctct gaaaacgaaa aaaccgcctt
     3541 gcagggcggt ttttcgaagg ttctctgagc taccaactct ttgaaccgag gtaactggct
     3601 tggaggagcg cagtcaccaa aacttgtcct ttcagtttag ccttaaccgg cgcatgactt
     3661 caagactaac tcctctaaat caattaccag tggctgctgc cagtggtgct tttgcatgtc
     3721 tttccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg gactgaacgg
     3781 ggggttcgtg catacagtcc agcttggagc gaactgccta cccggaactg agtgtcaggc
     3841 gtggaatgag acaaacgcgg ccataacagc ggaatgacac cggtaaaccg aaaggcagga
     3901 acaggagagc gcacgaggga gccgccaggg gaaacgcctg gtatctttat agtcctgtcg
     3961 ggtttcgcca ccactgattt gagcgtcaga tttcgtgatg cttgtcaggg gggcggagcc
     4021 tatggaaaaa cggctttgcc gcggccctct cacttccc
//
