LOCUS       sequence-12073-e        4587 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Entry vector for cloning  miR30-based shRNA driven by TRE promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4587)
  AUTHORS   Shin KJ, Wall EA, Zavzavadjian JR, Santat LA, Liu J, Hwang JI, 
            Rebres R, Roach T, Seaman W, Simon MI, Fraser ID
  TITLE     A single lentiviral vector platform for microRNA-based conditional 
            RNA interference and coordinated transgene expression.
  JOURNAL   Proc Natl Acad Sci U S A. 2006 Sep 12. 103(37):13759-64.
  PUBMED    16945906
REFERENCE   2  (bases 1 to 4587)
  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: "Proc Natl 
            Acad Sci U S A. 2006 Sep 12. 103(37):13759-64."
COMMENT     Sequence Label: pEN_TmiRc3
FEATURES             Location/Qualifiers
     source          1..4587
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     25..48
                     /label=pENTR-F
                     /note="pENTR vectors, forward primer"
     protein_bind    67..166
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     protein_bind    join(205..223,224..246,247..265,266..288,289..307,308..330,
                     331..349,350..372,373..391,392..414,415..433,434..456,
                     457..475)
                     /label=tetracycline response element
                     /bound_moiety="tetracycline repressor TetR"
                     /note="contains seven copies of the tetracycline operator 
                     tetO"
     promoter        508..546
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     543..567
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     ncRNA           709..830
                     /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"
     CDS             866..1171
                     /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="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRIVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     1050..1069
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     ncRNA           1192..1318
                     /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(1357..1456)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1568..1587)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(1646..1665)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     CDS             complement(2239..2772)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPRFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(2961..2966,2967..2983,2984..2989))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     rep_origin      3646..4234
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4135..4154
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      4399..4485
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      join(4577..4587,1..17)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
ORIGIN
        1 ctgacggatg gcctttttgc gtttctacaa actcttcctg ttagttagtt acttaagctc
       61 gggccccaaa taatgatttt attttgactg atagtgacct gttcgttgca acaaattgat
      121 aagcaatgct tttttataat gccaactttg tacaaaaaag caggctttaa aggaaccaat
      181 tcagtcgact ggatccttta ccactcccta tcagtgatag agaaaagtga aagtcgagtt
      241 taccactccc tatcagtgat agagaaaagt gaaagtcgag tttaccactc cctatcagtg
      301 atagagaaaa gtgaaagtcg agtttaccac tccctatcag tgatagagaa aagtgaaagt
      361 cgagtttacc actccctatc agtgatagag aaaagtgaaa gtcgagttta ccactcccta
      421 tcagtgatag agaaaagtga aagtcgagtt taccactccc tatcagtgat agagaaaagt
      481 gaaagtcgag ctcggtaccc gggtcgaggt aggcgtgtac ggtgggaggc ctatataagc
      541 agagctcgtt tagtgaaccg tcagatcgcc tggagacgcc atccacgctg ttttgacctc
      601 catagaagac accgggaccg atccagcctc cgcggccccg aactaggcct aggcgtctga
      661 tcactagtga ctctagtcct agtcgactag ggataacagg gtaattgttt gaatgaggct
      721 tcagtacttt acagaatcgt tgcctgcaca tcttggaaac acttgctggg attacttctt
      781 caggttaacc caacagaagg ctcgagaagg tatattgctg ttgacagtga gcgtgctgca
      841 ggtaagcttg aggtgtgctt ccagaatgca gtttaaggtt tacacctata aaagagagag
      901 ccgttatcgt ctgtttgtgg atgtacagag tgatattatt gacacgcccg ggcgacggat
      961 agtgatcccc ctggccagtg cacgtctgct gtcagataaa gtctcccgtg aactttaccc
     1021 ggtggtgcat atcggggatg aaagctggcg catgatgacc accgatatgg ccagtgtgcc
     1081 ggtctccgtt atcggggaag aagtggctga tctcagccac cgcgaaaatg acatcaaaaa
     1141 cgccattaac ctgatgttct ggggaatata gaattccaac ctgctgcttg cctactgcct
     1201 cggaattcaa ggggctactt taggagcaat tatcttgttt actaaaactg aataccttgc
     1261 tatctctttg atacattttt acaaagctga attaaaatgg tataaattaa atcacttttt
     1321 tcaattggaa gactaatgcg ttttcgagat atctagaccc agctttcttg tacaaagttg
     1381 gcattataag aaagcattgc ttatcaattt gttgcaacga acaggtcact atcagtcaaa
     1441 ataaaatcat tatttgccat ccagctgcag ctctggcccg tgtctcaaaa tctctgatgt
     1501 tacattgcac aagataaaaa tatatcatca tgaacaataa aactgtctgc ttacataaac
     1561 agtaatacaa ggggtgttat gagccatatt caacgggaaa cgtcgaggcc gcgattaaat
     1621 tccaacatgg atgctgattt atatgggtat aaatgggctc gcgataatgt cgggcaatca
     1681 ggtgcgacaa tctatcgctt gtatgggaag cccgatgcgc cagagttgtt tctgaaacat
     1741 ggcaaaggta gcgttgccaa tgatgttaca gatgagatgg tcagactaaa ctggctgacg
     1801 gaatttatgc ctcttccgac catcaagcat tttatccgta ctcctgatga tgcatggtta
     1861 ctcaccactg cgatccccgg aaaaacagca ttccaggtat tagaagaata tcctgattca
     1921 ggtgaaaata ttgttgatgc gctggcagtg tccctgcgcc ggttgcattc gattcctgtt
     1981 tgtaattgtc cttttaacag cgatcgcgta tttcgtctcg ctcaggcgca atcacgaatg
     2041 aataacggtt tggttgatgc gagtgatttt gatgacgagc gtaatggctg gcctgttgaa
     2101 caagtctgga aagaaatgca taaacttttg ccattctcac cggattcagt cgtcactcat
     2161 ggtgatttct cacttgataa ccttattttt gacgagggga aattaggccg ggaagccgat
     2221 ctcggcttga acgaattgtt aggtggcggt acttgggtcg atatcaaagt gcatcacttc
     2281 ttcccgtatg cccaactttg tatagagagc cactgcggga tcgtcaccgt aatctgcttg
     2341 cacgtagatc acataagcac caagcgcgtt ggcctcatgc ttgaggagat tgatgagcgc
     2401 ggtggcaatg ccctgcctcc ggtgctcgcc ggagactgcg agatcataga tatagatctc
     2461 actacgcggc tgctcaaacc tgggcagaac gtaagccgcg agagcgccaa caaccgcttc
     2521 ttggtcgaag gcagcaagcg cgatgaatgt cttactacgg agcaagttcc cgaggtaatc
     2581 ggagtccggc tgatgttggg agtaggtggc tacgtctccg aactcacgac cgaaaagatc
     2641 aagagcagcc cgcatggatt tgacttggtc agggccgagc ctacatgtgc gaatgatgcc
     2701 catacttgag ccacctaact ttgttttagg gcgactgccc tgctgcgtaa catcgttgct
     2761 gctgcgtaac atcgttgctg ctccataaca tcaaacatcg acccacggcg taacgcgctt
     2821 gctgcttgga tgcccgaggc atagactgta caaaaaaaca gtcataacaa gccatgaaaa
     2881 ccgccactgc gccgttacca ccgctgcgtt cggtcaaggt tctggaccag ttgcgtgagc
     2941 gcatacgcta cttgcattac agtttacgaa ccgaacaggc ttatgtcaac tgggttcgtg
     3001 ccttcatccg tttccacggt gtgcgtcacc cggcaacctt gggcagcagc gaagtcgagg
     3061 catttctgtc ctggctggcg aacgagcgca aggtttcggt ctccacgcat cgtcaggcat
     3121 tggcggcctt gctgttcttc tacggcaagg tgctgtgcac ggatctgccc tggcttcagg
     3181 agatcggaag acctcggccg tcgcggcgct tgccggtggt gctgaccccg gatgaagtgg
     3241 ttcgcatcct cggttttctg gaaggcgagc atcgtttgtt cgcccaggac tctagctata
     3301 gttctagtgg ttggctacta ataggttgta ttgatgttgg acgagtcgga atcgcagacc
     3361 gataccagga tcttgccatc ctatggaact gcctcggtga gttttctcct tcattacaga
     3421 aacggctttt tcaaaaatat ggtattgata atcctgatat gaataaattg cagtttcatt
     3481 tgatgctcga tgagtttttc taatcagaat tggttaattg gttgtaacac tggcagagca
     3541 ttacgctgac ttgacgggac ggcgcaagct catgaccaaa atcccttaac gtgagttttc
     3601 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     3661 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     3721 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     3781 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     3841 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     3901 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     3961 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     4021 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     4081 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     4141 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     4201 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     4261 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     4321 tgtggataac cgtattaccg ctagcatgga tctcggggac gtctaactac taagcgagag
     4381 tagggaactg ccaggcatca aataaaacga aaggctcagt cggaagactg ggcctttcgt
     4441 tttatctgtt gtttgtcggt gaacgctctc ctgagtagga caaatccgcc gggagcggat
     4501 ttgaacgttg tgaagcaacg gcccggaggg tggcgggcag gacgcccgcc ataaactgcc
     4561 aggcatcaaa ctaagcagaa ggccatc
//
