LOCUS       sequence-342059-        3678 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Entry vector for tightly controlled gene expression by a 
            tetracycline-responsive promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3678)
  AUTHORS   Araki M, Yoshimoto K, Ohta M, Katada T, Kontani K
  TITLE     Development of a versatile HPLC-based method to evaluate the 
            activation status of small GTPases.
  JOURNAL   J Biol Chem. 2021 Nov 18:101428. doi: 10.1016/j.jbc.2021.101428.
  PUBMED    34801548
REFERENCE   2  (bases 1 to 3678)
  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: "J Biol 
            Chem. 2021 Nov 18:101428. doi: 10.1016/j.jbc.2021.101428."
COMMENT     Sequence Label: pENTR_TiTRE
FEATURES             Location/Qualifiers
     source          1..3678
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        35..349
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    43..61
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    79..97
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    114..132
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    150..168
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    186..204
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    221..239
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    257..275
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     322..346
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     polyA_signal    484..1106
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(683..702)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     protein_bind    complement(1145..1244)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1261..1280)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1262..1280)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1285..1301)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1285..1301)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1403..1422)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1414..2223
                     /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     complement(1481..1500)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2370..2958
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2859..2878
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3112..3129
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(3288..3315)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(3407..3493)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     3542..3564
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3556..3573
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3557..3573
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    join(3589..3678,1..10)
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
ORIGIN
        1 aaagcaggct tcctttacca ctcgactgga tcccgagttt actccctatc agtgatagag
       61 aacgtatgtc gagtttactc cctatcagtg atagagaacg atgtcgagtt tactccctat
      121 cagtgataga gaacgtatgt cgagtttact ccctatcagt gatagagaac gtatgtcgag
      181 tttactccct atcagtgata gagaacgtat gtcgagttta tccctatcag tgatagagaa
      241 cgtatgtcga gtttactccc tatcagtgat agagaacgta tgtcgaggta ggcgtgtacg
      301 gtgggaggcc tatataagca gagctcgttt agtgaaccgt cagatcgcca ctagtcaaga
      361 caccgggacc gatccagcct ccgcggcccc gaactagtcc gtttagtgaa ccgtcagaat
      421 tcagatctgg taccacgcgt atcgataagc ttgcatgcct gcaggtcgac tctagaggat
      481 cccgggtggc atccctgtga cccctcccca gtgcctctcc tggccctgga agttgccact
      541 ccagtgccca ccagccttgt cctaataaaa ttaagttgca tcattttgtc tgactaggtg
      601 tccttctata atattatggg gtggaggggg gtggtatgga gcaaggggca agttgggaag
      661 acaacctgta gggcctgcgg ggtctattgg gaaccaagct ggagtgcagt ggcacaatct
      721 tggctcactg caatctccgc ctcctgggtt caagcgattc tcctgcctca gcctcccgag
      781 ttgttgggat tccaggcatg catgaccagg ctcagctaat ttttgttttt ttggtagaga
      841 cggggtttca ccatattggc caggctggtc tccaactcct aatctcaggt gatctaccca
      901 ccttggcctc ccaaattgct gggattacag gcgtgaacca ctgctccctt ccctgtcctt
      961 ctgattttaa aataactata ccagcaggag gacgtccaga cacagcatag gctacctggc
     1021 catgcccaac cggtgggaca tttgagttgc ttgcttggca ctgtcctctc atgcgttggg
     1081 tccactcagt agatgcctgt tgaattgggt acgcggccag cttggctgtg gaatgtgtgt
     1141 cagtacccag ctttcttgta caaagttggc attataagaa agcattgctt atcaatttgt
     1201 tgcaacgaac aggtcactat cagtcaaaat aaaatcatta tttgccatcc agctgatatc
     1261 ccctatagtg agtcgtatta catggtcata gctgtttcct ggcagctctg gcccgtgtct
     1321 caaaatctct gatgttacat tgcacaagat aaaaatatat catcatgaac aataaaactg
     1381 tctgcttaca taaacagtaa tacaaggggt gttatgagcc atattcaacg ggaaacgtcg
     1441 aggccgcgat taaattccaa catggatgct gatttatatg ggtataaatg ggctcgcgat
     1501 aatgtcgggc aatcaggtgc gacaatctat cgcttgtatg ggaagcccga tgcgccagag
     1561 ttgtttctga aacatggcaa aggtagcgtt gccaatgatg ttacagatga gatggtcaga
     1621 ctaaactggc tgacggaatt tatgcctctt ccgaccatca agcattttat ccgtactcct
     1681 gatgatgcat ggttactcac cactgcgatc cccggaaaaa cagcattcca ggtattagaa
     1741 gaatatcctg attcaggtga aaatattgtt gatgcgctgg cagtgttcct gcgccggttg
     1801 cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg tctcgctcag
     1861 gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg attttgatga cgagcgtaat
     1921 ggctggcctg ttgaacaagt ctggaaagaa atgcataaac ttttgccatt ctcaccggat
     1981 tcagtcgtca ctcatggtga tttctcactt gataacctta tttttgacga ggggaaatta
     2041 ataggttgta ttgatgttgg acgagtcgga atcgcagacc gataccagga tcttgccatc
     2101 ctatggaact gcctcggtga gttttctcct tcattacaga aacggctttt tcaaaaatat
     2161 ggtattgata atcctgatat gaataaattg cagtttcatt tgatgctcga tgagtttttc
     2221 taatcagaat tggttaattg gttgtaacac tggcagagca ttacgctgac ttgacgggac
     2281 ggcgcaagct catgaccaaa atcccttaac gtgagttacg cgtcgttcca ctgagcgtca
     2341 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     2401 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     2461 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     2521 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     2581 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     2641 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     2701 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     2761 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     2821 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     2881 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     2941 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     3001 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     3061 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     3121 gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg
     3181 attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac
     3241 gcaattaata cgcgtaccgc tagccaggaa gagtttgtag aaacgcaaaa aggccatccg
     3301 tcaggatggc cttctgctta gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc
     3361 accctccggg ccgttgcttc acaacgttca aatccgctcc cggcggattt gtcctactca
     3421 ggagagcgtt caccgacaaa caacagataa aacgaaaggc ccagtcttcc gactgagcct
     3481 ttcgttttat ttgatgcctg gcagttccct actctcgcgt taacgctagc atggatgttt
     3541 tcccagtcac gacgttgtaa aacgacggcc agtcttaagc tcgggcccca aataatgatt
     3601 ttattttgac tgatagtgac ctgttcgttg caacaaattg atgagcaatg cttttttata
     3661 atgccaactt tgtacaaa
//
