LOCUS       sequence-12072-e        4608 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Entry vector for cloning miR30-based shRNA driven by human U6 
            promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4608)
  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 4608)
  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_hUmiRc2
FEATURES             Location/Qualifiers
     source          1..4608
                     /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"
     promoter        426..675
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     426..446
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     598..617
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     ncRNA           730..851
                     /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             887..1192
                     /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     1071..1090
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     ncRNA           1213..1339
                     /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(1378..1477)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1589..1608)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(1667..1686)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     CDS             complement(2260..2793)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPRFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(2982..2987,2988..3004,3005..3010))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     rep_origin      3667..4255
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4156..4175
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      4420..4506
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      join(4598..4608,1..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 ggatcccccc gagtccaaca cccgtgggaa tcccatgggc accatggccc
      241 ctcgctccaa aaatgctttc gcgtctcgca gacactgctc ggtagtttcg gggatcagcg
      301 tttgagtaag agcccgcgtc tgaaccctcc gcgccgcccc ggcccagtgg aaagacgcgc
      361 aggcaaaacg caccacgtga cggagcgtga ccgcgcgccg agcgcgcgcc aaggtcgggc
      421 aggaagaggg cctatttccc atgattcctt catatttgca tatacgatac aaggctgtta
      481 gagagataat tagaattaat ttgactgtaa acacaaagat attagtacaa aatacgtgac
      541 gtagaaagta ataatttctt gggtagtttg cagtttttaa aattatgttt taaaatggac
      601 tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg
      661 aaaggacgaa acaccgtgct cgcttcggca gcacatatac tagtcgacta gggataacag
      721 ggtaattgtt tgaatgaggc ttcagtactt tacagaatcg ttgcctgcac atcttggaaa
      781 cacttgctgg gattacttct tcaggttaac ccaacagaag gctcgagaag gtatattgct
      841 gttgacagtg agcgtgctgc aggtaagctt gaggtgtgct tccagaatgc agtttaaggt
      901 ttacacctat aaaagagaga gccgttatcg tctgtttgtg gatgtacaga gtgatattat
      961 tgacacgccc gggcgacgga tagtgatccc cctggccagt gcacgtctgc tgtcagataa
     1021 agtctcccgt gaactttacc cggtggtgca tatcggggat gaaagctggc gcatgatgac
     1081 caccgatatg gccagtgtgc cggtctccgt tatcggggaa gaagtggctg atctcagcca
     1141 ccgcgaaaat gacatcaaaa acgccattaa cctgatgttc tggggaatat agaattccaa
     1201 cctgctgctt gcctactgcc tcggaattca aggggctact ttaggagcaa ttatcttgtt
     1261 tactaaaact gaataccttg ctatctcttt gatacatttt tacaaagctg aattaaaatg
     1321 gtataaatta aatcactttt ttcaattgga agactaatgc gttttcgaga tatctagacc
     1381 cagctttctt gtacaaagtt ggcattataa gaaagcattg cttatcaatt tgttgcaacg
     1441 aacaggtcac tatcagtcaa aataaaatca ttatttgcca tccagctgca gctctggccc
     1501 gtgtctcaaa atctctgatg ttacattgca caagataaaa atatatcatc atgaacaata
     1561 aaactgtctg cttacataaa cagtaataca aggggtgtta tgagccatat tcaacgggaa
     1621 acgtcgaggc cgcgattaaa ttccaacatg gatgctgatt tatatgggta taaatgggct
     1681 cgcgataatg tcgggcaatc aggtgcgaca atctatcgct tgtatgggaa gcccgatgcg
     1741 ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca atgatgttac agatgagatg
     1801 gtcagactaa actggctgac ggaatttatg cctcttccga ccatcaagca ttttatccgt
     1861 actcctgatg atgcatggtt actcaccact gcgatccccg gaaaaacagc attccaggta
     1921 ttagaagaat atcctgattc aggtgaaaat attgttgatg cgctggcagt gtccctgcgc
     1981 cggttgcatt cgattcctgt ttgtaattgt ccttttaaca gcgatcgcgt atttcgtctc
     2041 gctcaggcgc aatcacgaat gaataacggt ttggttgatg cgagtgattt tgatgacgag
     2101 cgtaatggct ggcctgttga acaagtctgg aaagaaatgc ataaactttt gccattctca
     2161 ccggattcag tcgtcactca tggtgatttc tcacttgata accttatttt tgacgagggg
     2221 aaattaggcc gggaagccga tctcggcttg aacgaattgt taggtggcgg tacttgggtc
     2281 gatatcaaag tgcatcactt cttcccgtat gcccaacttt gtatagagag ccactgcggg
     2341 atcgtcaccg taatctgctt gcacgtagat cacataagca ccaagcgcgt tggcctcatg
     2401 cttgaggaga ttgatgagcg cggtggcaat gccctgcctc cggtgctcgc cggagactgc
     2461 gagatcatag atatagatct cactacgcgg ctgctcaaac ctgggcagaa cgtaagccgc
     2521 gagagcgcca acaaccgctt cttggtcgaa ggcagcaagc gcgatgaatg tcttactacg
     2581 gagcaagttc ccgaggtaat cggagtccgg ctgatgttgg gagtaggtgg ctacgtctcc
     2641 gaactcacga ccgaaaagat caagagcagc ccgcatggat ttgacttggt cagggccgag
     2701 cctacatgtg cgaatgatgc ccatacttga gccacctaac tttgttttag ggcgactgcc
     2761 ctgctgcgta acatcgttgc tgctgcgtaa catcgttgct gctccataac atcaaacatc
     2821 gacccacggc gtaacgcgct tgctgcttgg atgcccgagg catagactgt acaaaaaaac
     2881 agtcataaca agccatgaaa accgccactg cgccgttacc accgctgcgt tcggtcaagg
     2941 ttctggacca gttgcgtgag cgcatacgct acttgcatta cagtttacga accgaacagg
     3001 cttatgtcaa ctgggttcgt gccttcatcc gtttccacgg tgtgcgtcac ccggcaacct
     3061 tgggcagcag cgaagtcgag gcatttctgt cctggctggc gaacgagcgc aaggtttcgg
     3121 tctccacgca tcgtcaggca ttggcggcct tgctgttctt ctacggcaag gtgctgtgca
     3181 cggatctgcc ctggcttcag gagatcggaa gacctcggcc gtcgcggcgc ttgccggtgg
     3241 tgctgacccc ggatgaagtg gttcgcatcc tcggttttct ggaaggcgag catcgtttgt
     3301 tcgcccagga ctctagctat agttctagtg gttggctact aataggttgt attgatgttg
     3361 gacgagtcgg aatcgcagac cgataccagg atcttgccat cctatggaac tgcctcggtg
     3421 agttttctcc ttcattacag aaacggcttt ttcaaaaata tggtattgat aatcctgata
     3481 tgaataaatt gcagtttcat ttgatgctcg atgagttttt ctaatcagaa ttggttaatt
     3541 ggttgtaaca ctggcagagc attacgctga cttgacggga cggcgcaagc tcatgaccaa
     3601 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     3661 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     3721 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     3781 tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca
     3841 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     3901 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     3961 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     4021 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     4081 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     4141 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     4201 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     4261 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     4321 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gctagcatgg atctcgggga
     4381 cgtctaacta ctaagcgaga gtagggaact gccaggcatc aaataaaacg aaaggctcag
     4441 tcggaagact gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct cctgagtagg
     4501 acaaatccgc cgggagcgga tttgaacgtt gtgaagcaac ggcccggagg gtggcgggca
     4561 ggacgcccgc cataaactgc caggcatcaa actaagcaga aggccatc
//
