LOCUS       sequence-12074-e        5303 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Entry vector for cloning miR30-based shRNA driven by TRE promoter 
            with GFP coexpression..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5303)
  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 5303)
  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_TGmiRc3
FEATURES             Location/Qualifiers
     source          1..5303
                     /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"
     CDS             join(659..661,662..664,665..1378)
                     /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(659..661,662..664,665..1375)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(704..725)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(965..984)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1312..1333
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     ncRNA           1425..1546
                     /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             1582..1887
                     /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     1766..1785
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     ncRNA           1908..2034
                     /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(2073..2172)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2284..2303)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(2362..2381)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     CDS             complement(2955..3488)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPRFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(3677..3682,3683..3699,3700..3705))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     rep_origin      4362..4950
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4851..4870
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      5115..5201
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      join(5293..5303,1..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 agtccaccat
      661 ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg
      721 cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg
      781 caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct
      841 cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca
      901 gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt
      961 caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt
     1021 gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa
     1081 gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg
     1141 catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga
     1201 ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta
     1261 cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct
     1321 gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtaaac
     1381 tagtgactct agtcctagtc gactagggat aacagggtaa ttgtttgaat gaggcttcag
     1441 tactttacag aatcgttgcc tgcacatctt ggaaacactt gctgggatta cttcttcagg
     1501 ttaacccaac agaaggctcg agaaggtata ttgctgttga cagtgagcgt gctgcaggta
     1561 agcttgaggt gtgcttccag aatgcagttt aaggtttaca cctataaaag agagagccgt
     1621 tatcgtctgt ttgtggatgt acagagtgat attattgaca cgcccgggcg acggatagtg
     1681 atccccctgg ccagtgcacg tctgctgtca gataaagtct cccgtgaact ttacccggtg
     1741 gtgcatatcg gggatgaaag ctggcgcatg atgaccaccg atatggccag tgtgccggtc
     1801 tccgttatcg gggaagaagt ggctgatctc agccaccgcg aaaatgacat caaaaacgcc
     1861 attaacctga tgttctgggg aatatagaat tccaacctgc tgcttgccta ctgcctcgga
     1921 attcaagggg ctactttagg agcaattatc ttgtttacta aaactgaata ccttgctatc
     1981 tctttgatac atttttacaa agctgaatta aaatggtata aattaaatca cttttttcaa
     2041 ttggaagact aatgcgtttt cgagatatct agacccagct ttcttgtaca aagttggcat
     2101 tataagaaag cattgcttat caatttgttg caacgaacag gtcactatca gtcaaaataa
     2161 aatcattatt tgccatccag ctgcagctct ggcccgtgtc tcaaaatctc tgatgttaca
     2221 ttgcacaaga taaaaatata tcatcatgaa caataaaact gtctgcttac ataaacagta
     2281 atacaagggg tgttatgagc catattcaac gggaaacgtc gaggccgcga ttaaattcca
     2341 acatggatgc tgatttatat gggtataaat gggctcgcga taatgtcggg caatcaggtg
     2401 cgacaatcta tcgcttgtat gggaagcccg atgcgccaga gttgtttctg aaacatggca
     2461 aaggtagcgt tgccaatgat gttacagatg agatggtcag actaaactgg ctgacggaat
     2521 ttatgcctct tccgaccatc aagcatttta tccgtactcc tgatgatgca tggttactca
     2581 ccactgcgat ccccggaaaa acagcattcc aggtattaga agaatatcct gattcaggtg
     2641 aaaatattgt tgatgcgctg gcagtgtccc tgcgccggtt gcattcgatt cctgtttgta
     2701 attgtccttt taacagcgat cgcgtatttc gtctcgctca ggcgcaatca cgaatgaata
     2761 acggtttggt tgatgcgagt gattttgatg acgagcgtaa tggctggcct gttgaacaag
     2821 tctggaaaga aatgcataaa cttttgccat tctcaccgga ttcagtcgtc actcatggtg
     2881 atttctcact tgataacctt atttttgacg aggggaaatt aggccgggaa gccgatctcg
     2941 gcttgaacga attgttaggt ggcggtactt gggtcgatat caaagtgcat cacttcttcc
     3001 cgtatgccca actttgtata gagagccact gcgggatcgt caccgtaatc tgcttgcacg
     3061 tagatcacat aagcaccaag cgcgttggcc tcatgcttga ggagattgat gagcgcggtg
     3121 gcaatgccct gcctccggtg ctcgccggag actgcgagat catagatata gatctcacta
     3181 cgcggctgct caaacctggg cagaacgtaa gccgcgagag cgccaacaac cgcttcttgg
     3241 tcgaaggcag caagcgcgat gaatgtctta ctacggagca agttcccgag gtaatcggag
     3301 tccggctgat gttgggagta ggtggctacg tctccgaact cacgaccgaa aagatcaaga
     3361 gcagcccgca tggatttgac ttggtcaggg ccgagcctac atgtgcgaat gatgcccata
     3421 cttgagccac ctaactttgt tttagggcga ctgccctgct gcgtaacatc gttgctgctg
     3481 cgtaacatcg ttgctgctcc ataacatcaa acatcgaccc acggcgtaac gcgcttgctg
     3541 cttggatgcc cgaggcatag actgtacaaa aaaacagtca taacaagcca tgaaaaccgc
     3601 cactgcgccg ttaccaccgc tgcgttcggt caaggttctg gaccagttgc gtgagcgcat
     3661 acgctacttg cattacagtt tacgaaccga acaggcttat gtcaactggg ttcgtgcctt
     3721 catccgtttc cacggtgtgc gtcacccggc aaccttgggc agcagcgaag tcgaggcatt
     3781 tctgtcctgg ctggcgaacg agcgcaaggt ttcggtctcc acgcatcgtc aggcattggc
     3841 ggccttgctg ttcttctacg gcaaggtgct gtgcacggat ctgccctggc ttcaggagat
     3901 cggaagacct cggccgtcgc ggcgcttgcc ggtggtgctg accccggatg aagtggttcg
     3961 catcctcggt tttctggaag gcgagcatcg tttgttcgcc caggactcta gctatagttc
     4021 tagtggttgg ctactaatag gttgtattga tgttggacga gtcggaatcg cagaccgata
     4081 ccaggatctt gccatcctat ggaactgcct cggtgagttt tctccttcat tacagaaacg
     4141 gctttttcaa aaatatggta ttgataatcc tgatatgaat aaattgcagt ttcatttgat
     4201 gctcgatgag tttttctaat cagaattggt taattggttg taacactggc agagcattac
     4261 gctgacttga cgggacggcg caagctcatg accaaaatcc cttaacgtga gttttcgttc
     4321 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     4381 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     4441 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     4501 aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     4561 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     4621 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     4681 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     4741 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     4801 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     4861 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     4921 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     4981 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     5041 gataaccgta ttaccgctag catggatctc ggggacgtct aactactaag cgagagtagg
     5101 gaactgccag gcatcaaata aaacgaaagg ctcagtcgga agactgggcc tttcgtttta
     5161 tctgttgttt gtcggtgaac gctctcctga gtaggacaaa tccgccggga gcggatttga
     5221 acgttgtgaa gcaacggccc ggagggtggc gggcaggacg cccgccataa actgccaggc
     5281 atcaaactaa gcagaaggcc atc
//
