LOCUS       sequence-12078-e        5153 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Entry vector for cloning miR30-based shRNA driven by TRE-tight 
            promoter with GFP coexpression..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5153)
  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 5153)
  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_TTGmiRc2
FEATURES             Location/Qualifiers
     source          1..5153
                     /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             join(524..526,527..529,530..1243)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(524..526,527..529,530..1240)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(569..590)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(830..849)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1177..1198
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     ncRNA           1275..1396
                     /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             1432..1737
                     /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     1616..1635
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     ncRNA           1758..1884
                     /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(1923..2022)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2134..2153)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(2212..2231)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     CDS             complement(2805..3338)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPRFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(3527..3532,3533..3549,3550..3555))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     rep_origin      4212..4800
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4701..4720
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      4965..5051
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      join(5143..5153,1..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 accatggtga gcaagggcga
      541 ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca
      601 caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa
      661 gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac
      721 ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa
      781 gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa
      841 ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct
      901 gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta
      961 caacagccac aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt
     1021 caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa
     1081 cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc
     1141 cgccctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac
     1201 cgccgccggg atcactctcg gcatggacga gctgtacaag taaactagtc gactagggat
     1261 aacagggtaa ttgtttgaat gaggcttcag tactttacag aatcgttgcc tgcacatctt
     1321 ggaaacactt gctgggatta cttcttcagg ttaacccaac agaaggctcg agaaggtata
     1381 ttgctgttga cagtgagcgt gctgcaggta agcttgaggt gtgcttccag aatgcagttt
     1441 aaggtttaca cctataaaag agagagccgt tatcgtctgt ttgtggatgt acagagtgat
     1501 attattgaca cgcccgggcg acggatagtg atccccctgg ccagtgcacg tctgctgtca
     1561 gataaagtct cccgtgaact ttacccggtg gtgcatatcg gggatgaaag ctggcgcatg
     1621 atgaccaccg atatggccag tgtgccggtc tccgttatcg gggaagaagt ggctgatctc
     1681 agccaccgcg aaaatgacat caaaaacgcc attaacctga tgttctgggg aatatagaat
     1741 tccaacctgc tgcttgccta ctgcctcgga attcaagggg ctactttagg agcaattatc
     1801 ttgtttacta aaactgaata ccttgctatc tctttgatac atttttacaa agctgaatta
     1861 aaatggtata aattaaatca cttttttcaa ttggaagact aatgcgtttt cgagatatct
     1921 agacccagct ttcttgtaca aagttggcat tataagaaag cattgcttat caatttgttg
     1981 caacgaacag gtcactatca gtcaaaataa aatcattatt tgccatccag ctgcagctct
     2041 ggcccgtgtc tcaaaatctc tgatgttaca ttgcacaaga taaaaatata tcatcatgaa
     2101 caataaaact gtctgcttac ataaacagta atacaagggg tgttatgagc catattcaac
     2161 gggaaacgtc gaggccgcga ttaaattcca acatggatgc tgatttatat gggtataaat
     2221 gggctcgcga taatgtcggg caatcaggtg cgacaatcta tcgcttgtat gggaagcccg
     2281 atgcgccaga gttgtttctg aaacatggca aaggtagcgt tgccaatgat gttacagatg
     2341 agatggtcag actaaactgg ctgacggaat ttatgcctct tccgaccatc aagcatttta
     2401 tccgtactcc tgatgatgca tggttactca ccactgcgat ccccggaaaa acagcattcc
     2461 aggtattaga agaatatcct gattcaggtg aaaatattgt tgatgcgctg gcagtgtccc
     2521 tgcgccggtt gcattcgatt cctgtttgta attgtccttt taacagcgat cgcgtatttc
     2581 gtctcgctca ggcgcaatca cgaatgaata acggtttggt tgatgcgagt gattttgatg
     2641 acgagcgtaa tggctggcct gttgaacaag tctggaaaga aatgcataaa cttttgccat
     2701 tctcaccgga ttcagtcgtc actcatggtg atttctcact tgataacctt atttttgacg
     2761 aggggaaatt aggccgggaa gccgatctcg gcttgaacga attgttaggt ggcggtactt
     2821 gggtcgatat caaagtgcat cacttcttcc cgtatgccca actttgtata gagagccact
     2881 gcgggatcgt caccgtaatc tgcttgcacg tagatcacat aagcaccaag cgcgttggcc
     2941 tcatgcttga ggagattgat gagcgcggtg gcaatgccct gcctccggtg ctcgccggag
     3001 actgcgagat catagatata gatctcacta cgcggctgct caaacctggg cagaacgtaa
     3061 gccgcgagag cgccaacaac cgcttcttgg tcgaaggcag caagcgcgat gaatgtctta
     3121 ctacggagca agttcccgag gtaatcggag tccggctgat gttgggagta ggtggctacg
     3181 tctccgaact cacgaccgaa aagatcaaga gcagcccgca tggatttgac ttggtcaggg
     3241 ccgagcctac atgtgcgaat gatgcccata cttgagccac ctaactttgt tttagggcga
     3301 ctgccctgct gcgtaacatc gttgctgctg cgtaacatcg ttgctgctcc ataacatcaa
     3361 acatcgaccc acggcgtaac gcgcttgctg cttggatgcc cgaggcatag actgtacaaa
     3421 aaaacagtca taacaagcca tgaaaaccgc cactgcgccg ttaccaccgc tgcgttcggt
     3481 caaggttctg gaccagttgc gtgagcgcat acgctacttg cattacagtt tacgaaccga
     3541 acaggcttat gtcaactggg ttcgtgcctt catccgtttc cacggtgtgc gtcacccggc
     3601 aaccttgggc agcagcgaag tcgaggcatt tctgtcctgg ctggcgaacg agcgcaaggt
     3661 ttcggtctcc acgcatcgtc aggcattggc ggccttgctg ttcttctacg gcaaggtgct
     3721 gtgcacggat ctgccctggc ttcaggagat cggaagacct cggccgtcgc ggcgcttgcc
     3781 ggtggtgctg accccggatg aagtggttcg catcctcggt tttctggaag gcgagcatcg
     3841 tttgttcgcc caggactcta gctatagttc tagtggttgg ctactaatag gttgtattga
     3901 tgttggacga gtcggaatcg cagaccgata ccaggatctt gccatcctat ggaactgcct
     3961 cggtgagttt tctccttcat tacagaaacg gctttttcaa aaatatggta ttgataatcc
     4021 tgatatgaat aaattgcagt ttcatttgat gctcgatgag tttttctaat cagaattggt
     4081 taattggttg taacactggc agagcattac gctgacttga cgggacggcg caagctcatg
     4141 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     4201 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     4261 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     4321 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta
     4381 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     4441 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     4501 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     4561 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     4621 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     4681 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     4741 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     4801 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     4861 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgctag catggatctc
     4921 ggggacgtct aactactaag cgagagtagg gaactgccag gcatcaaata aaacgaaagg
     4981 ctcagtcgga agactgggcc tttcgtttta tctgttgttt gtcggtgaac gctctcctga
     5041 gtaggacaaa tccgccggga gcggatttga acgttgtgaa gcaacggccc ggagggtggc
     5101 gggcaggacg cccgccataa actgccaggc atcaaactaa gcagaaggcc atc
//
