LOCUS       sequence-12077-e        4422 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Entry vector for cloning miR30-based shRNA driven by TRE-tight 
            promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4422)
  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 4422)
  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_TTmiRc2
FEATURES             Location/Qualifiers
     source          1..4422
                     /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        198..512
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    206..224
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    242..260
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    277..295
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    313..331
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    349..367
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    384..402
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    420..438
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     485..509
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     ncRNA           544..665
                     /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             701..1006
                     /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     885..904
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     ncRNA           1027..1153
                     /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(1192..1291)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1403..1422)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(1481..1500)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     CDS             complement(2074..2607)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPRFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(2796..2801,2802..2818,2819..2824))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     rep_origin      3481..4069
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3970..3989
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      4234..4320
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      join(4412..4422,1..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 ggatcccgag tttactccct atcagtgata gagaacgtat gtcgagttta
      241 ctccctatca gtgatagaga acgatgtcga gtttactccc tatcagtgat agagaacgta
      301 tgtcgagttt actccctatc agtgatagag aacgtatgtc gagtttactc cctatcagtg
      361 atagagaacg tatgtcgagt ttatccctat cagtgataga gaacgtatgt cgagtttact
      421 ccctatcagt gatagagaac gtatgtcgag gtaggcgtgt acggtgggag gcctatataa
      481 gcagagctcg tttagtgaac cgtcagatcg ccactagtcg actagggata acagggtaat
      541 tgtttgaatg aggcttcagt actttacaga atcgttgcct gcacatcttg gaaacacttg
      601 ctgggattac ttcttcaggt taacccaaca gaaggctcga gaaggtatat tgctgttgac
      661 agtgagcgtg ctgcaggtaa gcttgaggtg tgcttccaga atgcagttta aggtttacac
      721 ctataaaaga gagagccgtt atcgtctgtt tgtggatgta cagagtgata ttattgacac
      781 gcccgggcga cggatagtga tccccctggc cagtgcacgt ctgctgtcag ataaagtctc
      841 ccgtgaactt tacccggtgg tgcatatcgg ggatgaaagc tggcgcatga tgaccaccga
      901 tatggccagt gtgccggtct ccgttatcgg ggaagaagtg gctgatctca gccaccgcga
      961 aaatgacatc aaaaacgcca ttaacctgat gttctgggga atatagaatt ccaacctgct
     1021 gcttgcctac tgcctcggaa ttcaaggggc tactttagga gcaattatct tgtttactaa
     1081 aactgaatac cttgctatct ctttgataca tttttacaaa gctgaattaa aatggtataa
     1141 attaaatcac ttttttcaat tggaagacta atgcgttttc gagatatcta gacccagctt
     1201 tcttgtacaa agttggcatt ataagaaagc attgcttatc aatttgttgc aacgaacagg
     1261 tcactatcag tcaaaataaa atcattattt gccatccagc tgcagctctg gcccgtgtct
     1321 caaaatctct gatgttacat tgcacaagat aaaaatatat catcatgaac aataaaactg
     1381 tctgcttaca taaacagtaa tacaaggggt gttatgagcc atattcaacg ggaaacgtcg
     1441 aggccgcgat taaattccaa catggatgct gatttatatg ggtataaatg ggctcgcgat
     1501 aatgtcgggc aatcaggtgc gacaatctat cgcttgtatg ggaagcccga tgcgccagag
     1561 ttgtttctga aacatggcaa aggtagcgtt gccaatgatg ttacagatga gatggtcaga
     1621 ctaaactggc tgacggaatt tatgcctctt ccgaccatca agcattttat ccgtactcct
     1681 gatgatgcat ggttactcac cactgcgatc cccggaaaaa cagcattcca ggtattagaa
     1741 gaatatcctg attcaggtga aaatattgtt gatgcgctgg cagtgtccct gcgccggttg
     1801 cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg tctcgctcag
     1861 gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg attttgatga cgagcgtaat
     1921 ggctggcctg ttgaacaagt ctggaaagaa atgcataaac ttttgccatt ctcaccggat
     1981 tcagtcgtca ctcatggtga tttctcactt gataacctta tttttgacga ggggaaatta
     2041 ggccgggaag ccgatctcgg cttgaacgaa ttgttaggtg gcggtacttg ggtcgatatc
     2101 aaagtgcatc acttcttccc gtatgcccaa ctttgtatag agagccactg cgggatcgtc
     2161 accgtaatct gcttgcacgt agatcacata agcaccaagc gcgttggcct catgcttgag
     2221 gagattgatg agcgcggtgg caatgccctg cctccggtgc tcgccggaga ctgcgagatc
     2281 atagatatag atctcactac gcggctgctc aaacctgggc agaacgtaag ccgcgagagc
     2341 gccaacaacc gcttcttggt cgaaggcagc aagcgcgatg aatgtcttac tacggagcaa
     2401 gttcccgagg taatcggagt ccggctgatg ttgggagtag gtggctacgt ctccgaactc
     2461 acgaccgaaa agatcaagag cagcccgcat ggatttgact tggtcagggc cgagcctaca
     2521 tgtgcgaatg atgcccatac ttgagccacc taactttgtt ttagggcgac tgccctgctg
     2581 cgtaacatcg ttgctgctgc gtaacatcgt tgctgctcca taacatcaaa catcgaccca
     2641 cggcgtaacg cgcttgctgc ttggatgccc gaggcataga ctgtacaaaa aaacagtcat
     2701 aacaagccat gaaaaccgcc actgcgccgt taccaccgct gcgttcggtc aaggttctgg
     2761 accagttgcg tgagcgcata cgctacttgc attacagttt acgaaccgaa caggcttatg
     2821 tcaactgggt tcgtgccttc atccgtttcc acggtgtgcg tcacccggca accttgggca
     2881 gcagcgaagt cgaggcattt ctgtcctggc tggcgaacga gcgcaaggtt tcggtctcca
     2941 cgcatcgtca ggcattggcg gccttgctgt tcttctacgg caaggtgctg tgcacggatc
     3001 tgccctggct tcaggagatc ggaagacctc ggccgtcgcg gcgcttgccg gtggtgctga
     3061 ccccggatga agtggttcgc atcctcggtt ttctggaagg cgagcatcgt ttgttcgccc
     3121 aggactctag ctatagttct agtggttggc tactaatagg ttgtattgat gttggacgag
     3181 tcggaatcgc agaccgatac caggatcttg ccatcctatg gaactgcctc ggtgagtttt
     3241 ctccttcatt acagaaacgg ctttttcaaa aatatggtat tgataatcct gatatgaata
     3301 aattgcagtt tcatttgatg ctcgatgagt ttttctaatc agaattggtt aattggttgt
     3361 aacactggca gagcattacg ctgacttgac gggacggcgc aagctcatga ccaaaatccc
     3421 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     3481 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     3541 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     3601 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
     3661 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     3721 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     3781 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     3841 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     3901 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     3961 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     4021 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     4081 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     4141 gttatcccct gattctgtgg ataaccgtat taccgctagc atggatctcg gggacgtcta
     4201 actactaagc gagagtaggg aactgccagg catcaaataa aacgaaaggc tcagtcggaa
     4261 gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag taggacaaat
     4321 ccgccgggag cggatttgaa cgttgtgaag caacggcccg gagggtggcg ggcaggacgc
     4381 ccgccataaa ctgccaggca tcaaactaag cagaaggcca tc
//
