LOCUS       sequence-198825-        4526 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tet inducible microRNA and cDNA expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4526)
  TITLE     Wright lab backbones
REFERENCE   2  (bases 1 to 4526)
  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"
COMMENT     Sequence Label: pTRE-T2-miR-PURO
FEATURES             Location/Qualifiers
     source          1..4526
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     24..43
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        24..42
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    72..96
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     ncRNA           129..155
                     /label=5' miR-155
                     /note="sequence upstream of the precursor of mouse miR-155 
                     microRNA (Uva et al., 2013)"
                     /ncRNA_class="miRNA"
     ncRNA           197..238
                     /label=3' miR-155
                     /note="sequence downstream of the precursor of mouse 
                     miR-155 microRNA (Uva et al., 2013)"
                     /ncRNA_class="miRNA"
     protein_bind    complement(267..291)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    423..504
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     607..629
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(667..685)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        753..857
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(858..926,927..1718)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGPRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1076..1095)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     primer_bind     complement(1860..1880)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1865..2194
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2045..2180
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2287..2886
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(2287..2306)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2783..2803
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    3116..3197
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     3207..3226
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      3264..3852
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3753..3772
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4006..4023
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4140..4161
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4176..4181,4182..4199,4200..4206)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     promoter        join(4215..4526,1..3)
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    4223..4241
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4259..4277
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4294..4312
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4330..4348
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4366..4384
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4401..4419
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4437..4455
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     4502..4526
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
ORIGIN
        1 gcctggagaa ttcttatcga aattaatacg actcactata gggagtccca agctggctag
       61 ttaagctatc aacaagtttg tacaaaaaag caggctttaa agggaggtag tgagtcgacc
      121 agtggatcct ggaggcttgc tgaaggctgt atgctggaga ccgatcaatc gatcaatcga
      181 tcaatcggtc tctcaggaca caaggcctgt tactagcact cacatggaac aaatggccca
      241 gatctggccg cactcgagat atccagaccc agctttcttg tacaaagtgg ttgatctaga
      301 gggcccgcgg ctagaggatc ataatcagcc ataccacatt tgtagaggtt ttacttgctt
      361 taaaaaacct cccacacctc cccctgaacc tgaaacataa aatgaatgca attgttgttg
      421 ttaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
      481 caaataaagc atttttttca ctgccactag agttaagcca gccccgacac ccgccaacac
      541 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
      601 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac
      661 gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt
      721 agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
      781 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
      841 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
      901 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
      961 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     1021 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     1081 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
     1141 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     1201 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     1261 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     1321 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     1381 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     1441 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     1501 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     1561 ccggtgagcg tgggcctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     1621 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     1681 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     1741 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     1801 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccag ctagcctgtg
     1861 gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     1921 aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg
     1981 cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc
     2041 gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat
     2101 tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg
     2161 aggaggcttt tttggaggcc taggcttttg caaaaagctt gcatgcctgc aggtcggccg
     2221 ccacgaccgg tgccgccacc atcccctgac ccacgcccct gacccctcac aaggagacga
     2281 ccttccatga ccgagtacaa gcccacggtg cgcctcgcca cccgcgacga cgtcccccgg
     2341 gccgtacgca ccctcgccgc cgcgttcgcc gactaccccg ccacgcgcca caccgtcgac
     2401 ccggaccgcc acatcgagcg ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg
     2461 ctcgacatcg gcaaggtgtg ggtcgcggac gacggcgccg cggtggcggt ctggaccacg
     2521 ccggagagcg tcgaagcggg ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg
     2581 agcggttccc ggctggccgc gcagcaacag atggaaggcc tcctggcgcc gcaccggccc
     2641 aaggagcccg cgtggttcct ggccaccgtc ggcgtctcgc ccgaccacca gggcaagggt
     2701 ctgggcagcg ccgtcgtgct ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc
     2761 ttcctggaaa cctccgcgcc ccgcaacctc cccttctacg agcggctcgg cttcaccgtc
     2821 accgccgacg tcgaggtgcc cgaaggaccg cgcacctggt gcatgacccg caagcctggt
     2881 gcctgacgcc cgccccacga cccgcagcgc ccgaccgaaa ggagcgcacg accccatggc
     2941 tccgaccgaa gccgacccgg gcggccccgc cgaccccgca cccgcccccg aggcccaccg
     3001 actcaagagg atcataatca gccataccac atttgtagag gttttacttg ctttaaaaaa
     3061 cctcccacac ctccccctga acctgaaaca taaaatgaat gcaattgttg ttgttaactt
     3121 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     3181 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatctcggcc
     3241 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     3301 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     3361 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
     3421 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     3481 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     3541 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     3601 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     3661 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     3721 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     3781 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     3841 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     3901 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     3961 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
     4021 gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat
     4081 taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt
     4141 aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt
     4201 atgttgtgtg gctggagttt actccctatc agtgatagag aacgtatgtc gagtttactc
     4261 cctatcagtg atagagaacg atgtcgagtt tactccctat cagtgataga gaacgtatgt
     4321 cgagtttact ccctatcagt gatagagaac gtatgtcgag tttactccct atcagtgata
     4381 gagaacgtat gtcgagttta tccctatcag tgatagagaa cgtatgtcga gtttactccc
     4441 tatcagtgat agagaacgta tgtcgaggta ggcgtgtacg gtgggaggcc tatataagca
     4501 gagctcgttt agtgaaccgt cagatc
//
