LOCUS       sequence-89573-e        5407 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  GATEWAY cloning compatible conditional shRNA expression vector; TetO
            flanked H1 RNA gene promoter; Gentamycin resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5407)
  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 5407)
  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 III
FEATURES             Location/Qualifiers
     source          1..5407
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(104..123)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(182..201)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     CDS             complement(775..1308)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPRFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(1497..1502,1503..1519,1520..1525))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     rep_origin      2182..2770
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2671..2690
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(2905..2932)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(3024..3110)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     protein_bind    3152..3251
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     protein_bind    join(3304..3322,3323..3345,3346..3364,3365..3387,3388..3406,
                     3407..3429,3430..3448,3449..3471,3472..3490,3491..3513,
                     3514..3532,3533..3555,3556..3574)
                     /label=tetracycline response element
                     /bound_moiety="tetracycline repressor TetR"
                     /note="contains seven copies of the tetracycline operator 
                     tetO"
     primer_bind     3750..3769
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     protein_bind    3814..3832
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     intron          4230..4433
                     /label=EF-1-alpha intron A
                     /note="truncated intron upstream of the start codon of 
                     human EF-1-alpha"
     primer_bind     4390..4410
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             4496..5209
                     /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(4526..4554)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     protein_bind    complement(5241..5340)
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(5356..5373)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5356..5372)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5365..5387)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 ctgcagctct ggcccgtgtc tcaaaatctc tgatgttaca ttgcacaaga taaaaatata
       61 tcatcatgaa caataaaact gtctgcttac ataaacagta atacaagggg tgttatgagc
      121 catattcaac gggaaacgtc gaggccgcga ttaaattcca acatggatgc tgatttatat
      181 gggtataaat gggctcgcga taatgtcggg caatcaggtg cgacaatcta tcgcttgtat
      241 gggaagcccg atgcgccaga gttgtttctg aaacatggca aaggtagcgt tgccaatgat
      301 gttacagatg agatggtcag actaaactgg ctgacggaat ttatgcctct tccgaccatc
      361 aagcatttta tccgtactcc tgatgatgca tggttactca ccactgcgat ccccggaaaa
      421 acagcattcc aggtattaga agaatatcct gattcaggtg aaaatattgt tgatgcgctg
      481 gcagtgttcc tgcgccggtt gcattcgatt cctgtttgta attgtccttt taacagcgat
      541 cgcgtatttc gtctcgctca ggcgcaatca cgaatgaata acggtttggt tgatgcgagt
      601 gattttgatg acgagcgtaa tggctggcct gttgaacaag tctggaaaga aatgcataaa
      661 cttttgccat tctcaccgga ttcagtcgtc actcatggtg atttctcact tgataacctt
      721 atttttgacg aggggaaatt aggccgggaa gccgatctcg gcttgaacga attgttaggt
      781 ggcggtactt gggtcgatat caaagtgcat cacttcttcc cgtatgccca actttgtata
      841 gagagccact gcgggatcgt caccgtaatc tgcttgcacg tagatcacat aagcaccaag
      901 cgcgttggcc tcatgcttga ggagattgat gagcgcggtg gcaatgccct gcctccggtg
      961 ctcgccggag actgcgagat catagatata tatctcacta cgcggctgct caaacctggg
     1021 cagaacgtaa gccgcgagag cgccaacaac cgcttcttgg tcgaaggcag caagcgcgat
     1081 gaatgtctta ctacggagca agttcccgag gtaatcggag tccggctgat gttgggagta
     1141 ggtggctacg tctccgaact cacgaccgaa aagatcaaga gcagcccgca tggatttgac
     1201 ttggtcaggg ccgagcctac atgtgcgaat gatgcccata cttgagccac ctaactttgt
     1261 tttagggcga ctgccctgct gcgtaacatc gttgctgctg cgtaacatcg ttgctgctcc
     1321 ataacatcaa acatcgaccc acggcgtaac gcgcttgctg cttggatgcc cgaggcatag
     1381 actgtacaaa aaaacagtca taacaagcca tgaaaaccgc cactgcgccg ttaccaccgc
     1441 tgcgttcggt caaggttctg gaccagttgc gtgagcgcat acgctacttg cattacagtt
     1501 tacgaaccga acaggcttat gtcaactggg ttcgtgcctt catccgtttc cacggtgtgc
     1561 gtcacccggc aaccttgggc agcagcgaag tcgaggcatt tctgtcctgg ctggcgaacg
     1621 agcgcaaggt ttcggtctcc acgcatcgtc aggcattggc ggccttgctg ttcttctacg
     1681 gcaaggtgct gtgcacggat ctgccctggc ttcaggagat cggaagacct cggccgtcgc
     1741 ggcgcttgcc ggtggtgctg accccggatg aagtggttcg catcctcggt tttctggaag
     1801 gcgagcatcg tttgttcgcc caggactcta gctatagttc tagtggttgg ctactaatag
     1861 gttgtattga tgttggacga gtcggaatcg cagaccgata ccaggatctt gccatcctat
     1921 ggaactgcct cggtgagttt tctccttcat tacagaaacg gctttttcaa aaatatggta
     1981 ttgataatcc tgatatgaat aaattgcagt ttcatttgat gctcgatgag tttttctaat
     2041 cagaattggt taattggttg taacactggc agagcattac gctgacttga cgggacggcg
     2101 caagctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     2161 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     2221 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     2281 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta
     2341 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     2401 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     2461 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     2521 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     2581 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     2641 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     2701 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     2761 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     2821 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgctag
     2881 ccaggaagag tttgtagaaa cgcaaaaagg ccatccgtca ggatggcctt ctgcttagtt
     2941 tgatgcctgg cagtttatgg cgggcgtcct gcccgccacc ctccgggccg ttgcttcaca
     3001 acgttcaaat ccgctcccgg cggatttgtc ctactcagga gagcgttcac cgacaaacaa
     3061 cagataaaac gaaaggccca gtcttccgac tgagcctttc gttttatttg atgcctggca
     3121 gttccctact ctcgcgttat cagctggatg gcaaataatg attttatttt gactgatagt
     3181 gacctgttcg ttgcaacaaa ttgatgagca atgctttttt ataatgccaa ctttgtacaa
     3241 gaaagctggg tcggcgcgcc cacccttgga tcgatccggc cttgccggcc tcgagtttac
     3301 cactccctat cagtgataga gaaaagtgaa agtcgagttt accactccct atcagtgata
     3361 gagaaaagtg aaagtcgagt ttaccactcc ctatcagtga tagagaaaag tgaaagtcga
     3421 gtttaccact ccctatcagt gatagagaaa agtgaaagtc gagtttacca ctccctatca
     3481 gtgatagaga aaagtgaaag tcgagtttac cactccctat cagtgataga gaaaagtgaa
     3541 agtcgagttt accactccct atcagtgata gagaaaagtg aaagtcgagc tcggtacccg
     3601 ggctgcagga attcgaacgc tgacgtcatc aacccgctcc aaggaatcgc gggcccagtg
     3661 tcactaggcg ggaacaccca gcgcgcgtgc gccctggcag gaagatggct gtgagggaca
     3721 ggggagtggc gccctgcaat atttgcatgt cgctatgtgt tctgggaaat caccataaac
     3781 gtgaaatgtc tttggatttg ggaatcttat aagtccctat cagtgataga gatctgcaca
     3841 ctggtatttc ggtttttggg gccgcgggcg gcgacggggc ccgtgcgtcc cagcgcacat
     3901 gttcggcgag gcggggcctg cgagcgcggc caccgagaat cggacggggg tagtctcaag
     3961 ctggccggcc tgctctggtg cctggcctcg cgccgccgtg tatcgccccg ccctgggcgg
     4021 caaggctggc ccggtcggca ccagttgcgt gagcggaaag atggccgctt cccggccctg
     4081 ctgcagggag ctcaaaatgg aggacgcggc gctcgggaga gcgggcgggt gagtcaccca
     4141 cacaaaggaa aagggccttt ccgtcctcag ccgtcgcttc atgtgactcc acggagtacc
     4201 gggcgccgtc caggcacctc gattagttct cgagcttttg gagtacgtcg tctttaggtt
     4261 ggggggaggg gttttatgcg atggagtttc cccacactga gtgggtggag actgaagtta
     4321 ggccagcttg gcacttgatg taattctcct tggaatttgc cctttttgag tttggatctt
     4381 ggttcattct caagcctcag acagtggttc aaagtttttt tcttccattt caggtgtcgt
     4441 gaattacatt tgcttctgac acaactgtgt tcactagcaa cctcaaacag acaccatggg
     4501 taaaggagaa gaacttttca ctggagttgt cccaattctt gttgaattag atggtgatgt
     4561 taatgggcaa aaattctctg tcaggggaga gggtgaaggt gatgcaacat acggaaaact
     4621 tacccttaaa tttatttgca ctactgggaa gctacctgtt ccctggccaa cacttgtcac
     4681 tactttgact tatggtgtac aatgcttcgc aagataccca gatcatatga agcggcacga
     4741 cttcctcaag agcgccatgc ctgagggata cgtgcaggag aggaccatct tcttcaagga
     4801 cgacgggaac tacaagacac gtgctgaagt caagtttgag ggagacaccc tcgtcaacag
     4861 gatcgagctt aagggaatcg atttcaagga ggacggaaac atcctcggcc acaagttgga
     4921 atacaactac aactcccaca acgtatacat catggccgac aagcaaaaga acggcatcaa
     4981 agccaacttc aagacccgcc acaacatcga agacggcggc gtgcaactcg ctgatcatta
     5041 tcaacaaaat actccaattg gcgatggccc tgtcctttta ccagacaacc attacctgtc
     5101 ctatcaatct gccctttccc aagatcccca cggaaagaga gatcacatgg tccttcttga
     5161 gtttgttaca tctgctggga ttacacatgg catggatgaa ctatacaaag aattcccggg
     5221 aagcttgatc agggccgcgg agcctgcttt tttgtacaaa gttggcatta taaaaaagca
     5281 ttgctcatca atttgttgca acgaacaggt cactatcagt caaaataaaa tcattatttg
     5341 gggcccgagc ttaagactgg ccgtcgtttt acaacgtcgt gactgggaaa acatccatgc
     5401 tagcgtt
//
