LOCUS       sequence-89572-e        4533 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  GATEWAY cloning compatible conditional shRNA expression vector; TetO
            flanked H1 RNA gene promoter; Kanamycin resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4533)
  AUTHORS   Sigl R, Ploner C, Shivalingaiah G, Kofler R, Geley S
  TITLE     Development of a Multipurpose GATEWAY-Based Lentiviral 
            Tetracycline-Regulated Conditional RNAi System (GLTR).
  JOURNAL   PLoS One. 2014 May 19;9(5):e97764. doi: 
            10.1371/journal.pone.0097764. eCollection 2014.
  PUBMED    24841113
REFERENCE   2  (bases 1 to 4533)
  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: "PLoS One.";
            date: "2014-05-19"; volume: "9(5):e97764. doi"; pages: " 
            10.1371/journal.pone.0097764. eCollection 2014"
COMMENT     Sequence Label: pENTR-THT
FEATURES             Location/Qualifiers
     source          1..4533
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    join(14..32,33..55,56..74,75..97,98..116,117..139,140..158,
                     159..181,182..200,201..223,224..242,243..265,266..284)
                     /label=tetracycline response element
                     /bound_moiety="tetracycline repressor TetR"
                     /note="contains seven copies of the tetracycline operator 
                     tetO"
     primer_bind     460..479
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     protein_bind    524..542
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     intron          940..1143
                     /label=EF-1-alpha intron A
                     /note="truncated intron upstream of the start codon of 
                     human EF-1-alpha"
     primer_bind     1100..1120
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             1206..1919
                     /codon_start=1
                     /gene="mgfp5"
                     /product="GFP with folding enhancement mutations"
                     /label=mgfp5
                     /note="suitable for expression in plants due to removal of 
                     a cryptic intron"
                     /translation="MGKGEELFTGVVPILVELDGDVNGQKFSVRGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKRHDFLKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKA
                     NFKTRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSQDPHGKRDHMVLLE
                     FVTSAGITHGMDELYK"
     primer_bind     complement(1236..1264)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     protein_bind    complement(1951..2050)
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2066..2083)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2066..2082)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2075..2097)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     terminator      2146..2232
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2324..2351
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     complement(2510..2527)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2681..3269)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2761..2780)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3416..4225)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     4139..4158
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4217..4236
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     4338..4354
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4338..4354
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     4359..4378
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4359..4377
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4395..4494
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
ORIGIN
        1 tcgagtttac cactccctat cagtgataga gaaaagtgaa agtcgagttt accactccct
       61 atcagtgata gagaaaagtg aaagtcgagt ttaccactcc ctatcagtga tagagaaaag
      121 tgaaagtcga gtttaccact ccctatcagt gatagagaaa agtgaaagtc gagtttacca
      181 ctccctatca gtgatagaga aaagtgaaag tcgagtttac cactccctat cagtgataga
      241 gaaaagtgaa agtcgagttt accactccct atcagtgata gagaaaagtg aaagtcgagc
      301 tcggtacccg ggctgcagga attcgaacgc tgacgtcatc aacccgctcc aaggaatcgc
      361 gggcccagtg tcactaggcg ggaacaccca gcgcgcgtgc gccctggcag gaagatggct
      421 gtgagggaca ggggagtggc gccctgcaat atttgcatgt cgctatgtgt tctgggaaat
      481 caccataaac gtgaaatgtc tttggatttg ggaatcttat aagtccctat cagtgataga
      541 gatctgcaca ctggtatttc ggtttttggg gccgcgggcg gcgacggggc ccgtgcgtcc
      601 cagcgcacat gttcggcgag gcggggcctg cgagcgcggc caccgagaat cggacggggg
      661 tagtctcaag ctggccggcc tgctctggtg cctggcctcg cgccgccgtg tatcgccccg
      721 ccctgggcgg caaggctggc ccggtcggca ccagttgcgt gagcggaaag atggccgctt
      781 cccggccctg ctgcagggag ctcaaaatgg aggacgcggc gctcgggaga gcgggcgggt
      841 gagtcaccca cacaaaggaa aagggccttt ccgtcctcag ccgtcgcttc atgtgactcc
      901 acggagtacc gggcgccgtc caggcacctc gattagttct cgagcttttg gagtacgtcg
      961 tctttaggtt ggggggaggg gttttatgcg atggagtttc cccacactga gtgggtggag
     1021 actgaagtta ggccagcttg gcacttgatg taattctcct tggaatttgc cctttttgag
     1081 tttggatctt ggttcattct caagcctcag acagtggttc aaagtttttt tcttccattt
     1141 caggtgtcgt gaattacatt tgcttctgac acaactgtgt tcactagcaa cctcaaacag
     1201 acaccatggg taaaggagaa gaacttttca ctggagttgt cccaattctt gttgaattag
     1261 atggtgatgt taatgggcaa aaattctctg tcaggggaga gggtgaaggt gatgcaacat
     1321 acggaaaact tacccttaaa tttatttgca ctactgggaa gctacctgtt ccctggccaa
     1381 cacttgtcac tactttgact tatggtgtac aatgcttcgc aagataccca gatcatatga
     1441 agcggcacga cttcctcaag agcgccatgc ctgagggata cgtgcaggag aggaccatct
     1501 tcttcaagga cgacgggaac tacaagacac gtgctgaagt caagtttgag ggagacaccc
     1561 tcgtcaacag gatcgagctt aagggaatcg atttcaagga ggacggaaac atcctcggcc
     1621 acaagttgga atacaactac aactcccaca acgtatacat catggccgac aagcaaaaga
     1681 acggcatcaa agccaacttc aagacccgcc acaacatcga agacggcggc gtgcaactcg
     1741 ctgatcatta tcaacaaaat actccaattg gcgatggccc tgtcctttta ccagacaacc
     1801 attacctgtc ctatcaatct gccctttccc aagatcccca cggaaagaga gatcacatgg
     1861 tccttcttga gtttgttaca tctgctggga ttacacatgg catggatgaa ctatacaaag
     1921 aattcccggg aagcttgatc agggccgcgg agcctgcttt tttgtacaaa gttggcatta
     1981 taaaaaagca ttgctcatca atttgttgca acgaacaggt cactatcagt caaaataaaa
     2041 tcattatttg gggcccgagc ttaagactgg ccgtcgtttt acaacgtcgt gactgggaaa
     2101 acatccatgc tagcgttaac gcgagagtag ggaactgcca ggcatcaaat aaaacgaaag
     2161 gctcagtcgg aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa cgctctcctg
     2221 agtaggacaa atccgccggg agcggatttg aacgttgtga agcaacggcc cggagggtgg
     2281 cgggcaggac gcccgccata aactgccagg catcaaacta agcagaaggc catcctgacg
     2341 gatggccttt ttgcgtttct acaaactctt cctggctagc ggtacgcgta ttaattgcgt
     2401 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     2461 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     2521 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     2581 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     2641 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     2701 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     2761 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     2821 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcaatgct
     2881 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     2941 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     3001 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     3061 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     3121 ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     3181 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     3241 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     3301 acgctcagtg gaacgacgcg taactcacgt taagggattt tggtcatgag cttgcgccgt
     3361 cccgtcaagt cagcgtaatg ctctgccagt gttacaacca attaaccaat tctgattaga
     3421 aaaactcatc gagcatcaaa tgaaactgca atttattcat atcaggatta tcaataccat
     3481 atttttgaaa aagccgtttc tgtaatgaag gagaaaactc accgaggcag ttccatagga
     3541 tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc aacatcaata caacctatta
     3601 atttcccctc gtcaaaaata aggttatcaa gtgagaaatc accatgagtg acgactgaat
     3661 ccggtgagaa tggcaaaagt ttatgcattt ctttccagac ttgttcaaca ggccagccat
     3721 tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct
     3781 gagcgagacg aaatacgcga tcgctgttaa aaggacaatt acaaacagga atcgaatgca
     3841 accggcgcag gaacactgcc agcgcatcaa caatattttc acctgaatca ggatattctt
     3901 ctaatacctg gaatgctgtt tttccgggga tcgcagtggt gagtaaccat gcatcatcag
     3961 gagtacggat aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc cagtttagtc
     4021 tgaccatctc atctgtaaca tcattggcaa cgctaccttt gccatgtttc agaaacaact
     4081 ctggcgcatc gggcttccca tacaagcgat agattgtcgc acctgattgc ccgacattat
     4141 cgcgagccca tttataccca tataaatcag catccatgtt ggaatttaat cgcggcctcg
     4201 acgtttcccg ttgaatatgg ctcataacac cccttgtatt actgtttatg taagcagaca
     4261 gttttattgt tcatgatgat atatttttat cttgtgcaat gtaacatcag agattttgag
     4321 acacgggcca gagctgccag gaaacagcta tgaccatgta atacgactca ctatagggga
     4381 tatcagctgg atggcaaata atgattttat tttgactgat agtgacctgt tcgttgcaac
     4441 aaattgatga gcaatgcttt tttataatgc caactttgta caagaaagct gggtcggcgc
     4501 gcccaccctt ggatcgatcc ggccttgccg gcc
//
