LOCUS       sequence-12079-e        5180 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Entry vector for cloning miR30-based shRNA driven by TRE-tight 
            promoter with dsRed2 coexpression..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5180)
  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 5180)
  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_TTRmiRc2
FEATURES             Location/Qualifiers
     source          1..5180
                     /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"
     CDS             524..1198
                     /codon_start=1
                     /product="improved tetrameric variant of DsRed fluorescent 
                     protein"
                     /label=DsRed2
                     /note="mammalian codon-optimized"
                     /translation="MASSENVITEFMRFKVRMEGTVNGHEFEIEGEGEGRPYEGHNTVK
                     LKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGV
                     ATVTQDSSLQDGCFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGETHK
                     ALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDAKLDITSHNEDYTIVEQYERTEGRHH
                     LFL"
     primer_bind     complement(704..724)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     1116..1139
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     misc_feature    1199..1255
                     /label=MCS
                     /note="multiple cloning site"
     ncRNA           1302..1423
                     /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             1459..1764
                     /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     1643..1662
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     ncRNA           1785..1911
                     /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(1950..2049)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2161..2180)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(2239..2258)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     CDS             complement(2832..3365)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPRFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(3554..3559,3560..3576,3577..3582))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     rep_origin      4239..4827
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4728..4747
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      4992..5078
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      join(5170..5180,1..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 ccactagtcc accatggcct cctccgagaa
      541 cgtcatcacc gagttcatgc gcttcaaggt gcgcatggag ggcaccgtga acggccacga
      601 gttcgagatc gagggcgagg gcgagggccg cccctacgag ggccacaaca ccgtgaagct
      661 gaaggtgacc aagggcggcc ccctgccctt cgcctgggac atcctgtccc cccagttcca
      721 gtacggctcc aaggtgtacg tgaagcaccc cgccgacatc cccgactaca agaagctgtc
      781 cttccccgag ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg gcgtggcgac
      841 cgtgacccag gactcctccc tgcaggacgg ctgcttcatc tacaaggtga agttcatcgg
      901 cgtgaacttc ccctccgacg gccccgtgat gcagaagaag accatgggct gggaggcctc
      961 caccgagcgc ctgtaccccc gcgacggcgt gctgaagggc gagacccaca aggccctgaa
     1021 gctgaaggac ggcggccact acctggtgga gttcaagtcc atctacatgg ccaagaagcc
     1081 cgtgcagctg cccggctact actacgtgga cgccaagctg gacatcacct cccacaacga
     1141 ggactacacc atcgtggagc agtacgagcg caccgagggc cgccaccacc tgttcctgag
     1201 atctcgagct caagcttcga attctgcagt cgacggtacc gcgggcccgg gatccaccgg
     1261 atctagataa actagtcgac tagggataac agggtaattg tttgaatgag gcttcagtac
     1321 tttacagaat cgttgcctgc acatcttgga aacacttgct gggattactt cttcaggtta
     1381 acccaacaga aggctcgaga aggtatattg ctgttgacag tgagcgtgct gcaggtaagc
     1441 ttgaggtgtg cttccagaat gcagtttaag gtttacacct ataaaagaga gagccgttat
     1501 cgtctgtttg tggatgtaca gagtgatatt attgacacgc ccgggcgacg gatagtgatc
     1561 cccctggcca gtgcacgtct gctgtcagat aaagtctccc gtgaacttta cccggtggtg
     1621 catatcgggg atgaaagctg gcgcatgatg accaccgata tggccagtgt gccggtctcc
     1681 gttatcgggg aagaagtggc tgatctcagc caccgcgaaa atgacatcaa aaacgccatt
     1741 aacctgatgt tctggggaat atagaattcc aacctgctgc ttgcctactg cctcggaatt
     1801 caaggggcta ctttaggagc aattatcttg tttactaaaa ctgaatacct tgctatctct
     1861 ttgatacatt tttacaaagc tgaattaaaa tggtataaat taaatcactt ttttcaattg
     1921 gaagactaat gcgttttcga gatatctaga cccagctttc ttgtacaaag ttggcattat
     1981 aagaaagcat tgcttatcaa tttgttgcaa cgaacaggtc actatcagtc aaaataaaat
     2041 cattatttgc catccagctg cagctctggc ccgtgtctca aaatctctga tgttacattg
     2101 cacaagataa aaatatatca tcatgaacaa taaaactgtc tgcttacata aacagtaata
     2161 caaggggtgt tatgagccat attcaacggg aaacgtcgag gccgcgatta aattccaaca
     2221 tggatgctga tttatatggg tataaatggg ctcgcgataa tgtcgggcaa tcaggtgcga
     2281 caatctatcg cttgtatggg aagcccgatg cgccagagtt gtttctgaaa catggcaaag
     2341 gtagcgttgc caatgatgtt acagatgaga tggtcagact aaactggctg acggaattta
     2401 tgcctcttcc gaccatcaag cattttatcc gtactcctga tgatgcatgg ttactcacca
     2461 ctgcgatccc cggaaaaaca gcattccagg tattagaaga atatcctgat tcaggtgaaa
     2521 atattgttga tgcgctggca gtgtccctgc gccggttgca ttcgattcct gtttgtaatt
     2581 gtccttttaa cagcgatcgc gtatttcgtc tcgctcaggc gcaatcacga atgaataacg
     2641 gtttggttga tgcgagtgat tttgatgacg agcgtaatgg ctggcctgtt gaacaagtct
     2701 ggaaagaaat gcataaactt ttgccattct caccggattc agtcgtcact catggtgatt
     2761 tctcacttga taaccttatt tttgacgagg ggaaattagg ccgggaagcc gatctcggct
     2821 tgaacgaatt gttaggtggc ggtacttggg tcgatatcaa agtgcatcac ttcttcccgt
     2881 atgcccaact ttgtatagag agccactgcg ggatcgtcac cgtaatctgc ttgcacgtag
     2941 atcacataag caccaagcgc gttggcctca tgcttgagga gattgatgag cgcggtggca
     3001 atgccctgcc tccggtgctc gccggagact gcgagatcat agatatagat ctcactacgc
     3061 ggctgctcaa acctgggcag aacgtaagcc gcgagagcgc caacaaccgc ttcttggtcg
     3121 aaggcagcaa gcgcgatgaa tgtcttacta cggagcaagt tcccgaggta atcggagtcc
     3181 ggctgatgtt gggagtaggt ggctacgtct ccgaactcac gaccgaaaag atcaagagca
     3241 gcccgcatgg atttgacttg gtcagggccg agcctacatg tgcgaatgat gcccatactt
     3301 gagccaccta actttgtttt agggcgactg ccctgctgcg taacatcgtt gctgctgcgt
     3361 aacatcgttg ctgctccata acatcaaaca tcgacccacg gcgtaacgcg cttgctgctt
     3421 ggatgcccga ggcatagact gtacaaaaaa acagtcataa caagccatga aaaccgccac
     3481 tgcgccgtta ccaccgctgc gttcggtcaa ggttctggac cagttgcgtg agcgcatacg
     3541 ctacttgcat tacagtttac gaaccgaaca ggcttatgtc aactgggttc gtgccttcat
     3601 ccgtttccac ggtgtgcgtc acccggcaac cttgggcagc agcgaagtcg aggcatttct
     3661 gtcctggctg gcgaacgagc gcaaggtttc ggtctccacg catcgtcagg cattggcggc
     3721 cttgctgttc ttctacggca aggtgctgtg cacggatctg ccctggcttc aggagatcgg
     3781 aagacctcgg ccgtcgcggc gcttgccggt ggtgctgacc ccggatgaag tggttcgcat
     3841 cctcggtttt ctggaaggcg agcatcgttt gttcgcccag gactctagct atagttctag
     3901 tggttggcta ctaataggtt gtattgatgt tggacgagtc ggaatcgcag accgatacca
     3961 ggatcttgcc atcctatgga actgcctcgg tgagttttct ccttcattac agaaacggct
     4021 ttttcaaaaa tatggtattg ataatcctga tatgaataaa ttgcagtttc atttgatgct
     4081 cgatgagttt ttctaatcag aattggttaa ttggttgtaa cactggcaga gcattacgct
     4141 gacttgacgg gacggcgcaa gctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     4201 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     4261 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     4321 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     4381 actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     4441 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     4501 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     4561 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     4621 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     4681 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     4741 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     4801 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     4861 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     4921 aaccgtatta ccgctagcat ggatctcggg gacgtctaac tactaagcga gagtagggaa
     4981 ctgccaggca tcaaataaaa cgaaaggctc agtcggaaga ctgggccttt cgttttatct
     5041 gttgtttgtc ggtgaacgct ctcctgagta ggacaaatcc gccgggagcg gatttgaacg
     5101 ttgtgaagca acggcccgga gggtggcggg caggacgccc gccataaact gccaggcatc
     5161 aaactaagca gaaggccatc
//
