LOCUS       sequence-218023-        4524 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 4524)
  TITLE     Wright lab backbones
REFERENCE   2  (bases 1 to 4524)
  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..4524
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    877..898
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(913..918,919..936,937..943)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     promoter        952..1266
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    960..978
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    996..1014
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1031..1049
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1067..1085
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1103..1121
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1138..1156
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1174..1192
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     1239..1263
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     primer_bind     1287..1306
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1287..1305
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1335..1359
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     ncRNA           1392..1418
                     /label=5' miR-155
                     /note="sequence upstream of the precursor of mouse miR-155 
                     microRNA (Uva et al., 2013)"
                     /ncRNA_class="miRNA"
     ncRNA           1460..1501
                     /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(1530..1554)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    1686..1767
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     1870..1892
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1930..1948)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2016..2120
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2121..2189,2190..2981)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGARGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2339..2358)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     primer_bind     complement(3122..3142)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3127..3456
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3307..3442
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3549..4148
                     /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(3549..3568)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4045..4065
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    4378..4459
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     4469..4488
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      841 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
      901 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggct ggagtttact
      961 ccctatcagt gatagagaac gtatgtcgag tttactccct atcagtgata gagaacgatg
     1021 tcgagtttac tccctatcag tgatagagaa cgtatgtcga gtttactccc tatcagtgat
     1081 agagaacgta tgtcgagttt actccctatc agtgatagag aacgtatgtc gagtttatcc
     1141 ctatcagtga tagagaacgt atgtcgagtt tactccctat cagtgataga gaacgtatgt
     1201 cgaggtaggc gtgtacggtg ggaggcctat ataagcagag ctcgtttagt gaaccgtcag
     1261 atcgcctgga gaattcttat cgaaattaat acgactcact atagggagtc ccaagctggc
     1321 tagttaagct atcaacaagt ttgtacaaaa aagcaggctt taaagggagg tagtgagtcg
     1381 accagtggat cctggaggct tgctgaaggc tgtatgctgg agaccgatca atcgatcaat
     1441 cgatcaatcg gtctctcagg acacaaggcc tgttactagc actcacatgg aacaaatggc
     1501 ccagatctgg ccgcactcga gatatccaga cccagctttc ttgtacaaag tggttgatct
     1561 agagggcccg cggctagagg atcataatca gccataccac atttgtagag gttttacttg
     1621 ctttaaaaaa cctcccacac ctccccctga acctgaaaca taaaatgaat gcaattgttg
     1681 ttgttaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     1741 tcacaaataa agcatttttt tcactgccac tagagttaag ccagccccga cacccgccaa
     1801 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     1861 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     1921 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
     1981 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     2041 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     2101 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     2161 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     2221 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     2281 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     2341 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     2401 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     2461 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     2521 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     2581 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     2641 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
     2701 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     2761 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     2821 gagccggtga gcgtggggct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     2881 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     2941 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     3001 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     3061 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cagctagctg
     3121 tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     3181 caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca
     3241 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact
     3301 ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta
     3361 atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag
     3421 tgaggaggct tttttggagg cctaggcttt tgcaaaaagc ttgcatgcct gcaggtcggc
     3481 cgccacgacc ggtgccgcca ccatcccctg acccacgccc ctgacccctc acaaggagac
     3541 gaccttccat gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac gacgtccccc
     3601 gggctgtacg caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg
     3661 acccggaccg ccacatcgag cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg
     3721 ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca
     3781 cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc atggccgagt
     3841 tgagcggttc ccggctggcc gcgcagcaac agatggaagg cctcctggcg ccgcaccggc
     3901 ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac cagggcaagg
     3961 gtctgggcag cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg
     4021 ccttcctgga aacctccgcg ccccgcaacc tccccttcta cgagcggctc ggcttcaccg
     4081 tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg
     4141 gtgcctgacg cccgccccac gacccgcagc gcccgaccga aaggagcgca cgaccccatg
     4201 gctccgaccg aagccgaccc gggcggcccc gccgaccccg cacccgcccc cgaggcccac
     4261 cgactcaaga ggatcataat cagccatacc acatttgtag aggttttact tgctttaaaa
     4321 aacctcccac acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac
     4381 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     4441 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatctggc
     4501 cgtagaaaag atcaaaggat cttc
//
