LOCUS       sequence-266811-        5173 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid contains the floxed TetOn3G-transactivator sequence for 
            expression of TetOn3G in the presence of Cre recombinase..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5173)
  AUTHORS   Heumuller M, Glock C, Rangaraju V, Biever A, Schuman EM
  TITLE     A genetically encodable cell-type-specific protein synthesis 
            inhibitor.
  JOURNAL   Nat Methods. 2019 Jul 15. pii: 10.1038/s41592-019-0468-x. doi: 
            10.1038/s41592-019-0468-x.
  PUBMED    31308551
REFERENCE   2  (bases 1 to 5173)
  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: "Nat 
            Methods. 2019 Jul 15. pii: 10.1038/s41592-019-0468-x. doi: 
            10.1038/s41592-019-0468-x."
COMMENT     Sequence Label: pCMV-FLEx-TetOn3G-3xHA-FLEx
FEATURES             Location/Qualifiers
     source          1..5173
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        286..665
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        666..869
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     820..840
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     914..933
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        914..932
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    978..1011
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    1074..1107
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(1123..1149)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(1156..1182)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(1186..1212)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(1222..1965)
                     /codon_start=1
                     /product="modified rtTA protein that binds tightly to 
                     promoters containing the tet operator in the presence of 
                     doxycycline"
                     /label=Tet-On(R) 3G
                     /translation="MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHSCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLKQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     protein_bind    complement(1969..2002)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    complement(2073..2106)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(2121..2138)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2127..2351
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   2436..2576
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2436..2565
                     /label=AAV2 ITR
     rep_origin      2651..3106
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2738..2757)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2948..2969
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3123..3142)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3242..3264
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3302..3320)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3388..3492
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3493..3561,3562..4353)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3711..3730)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4524..5112
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5013..5032
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggcaattc agtcgataac tataacggtc ctaaggtagc gatttaaata
      181 cgcgctctct taaggtagcc ccgggacgcg tcaattgcat gaagaatctg cttagggtta
      241 ggcgttttgc gctgcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga
      301 ctagttatta atagtaatca attacggggt cattagttca tagcccatat atggagttcc
      361 gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat
      421 tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc
      481 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
      541 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt
      601 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
      661 ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg
      721 gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac
      781 gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg
      841 tacggtggga ggtctatata agcagagctc tctggctaac tagagaaccc actgcttact
      901 ggcttatcga aattaatacg actcactata gggagaccca agctggctag cgtttaaact
      961 taagcttggt accgagcata acttcgtata aagtatccta tacgaagtta tatcaaaata
     1021 ggaagaccaa tgcttcacca tcgacccgaa ttgccaagca tcaccatcga cccataactt
     1081 cgtataatgt atgctatacg aagttatact tcatctagaa ccagcgtaat ctggaacgtc
     1141 ataaggatag gctcctgcat agtccggcac gtcatacggg tagcccgcat agtcaggaac
     1201 atcgtatggg tatccgcttc ccccggggag catgtcaagg tcaaaatcgt caagagcgtc
     1261 agcaggcagc atatcaaggt caaagtcgtc aagggcatcg gctgggagca tgtctaagtc
     1321 aaaatcgtca agggcgtcgg tcggcccgcc gctttcgcac tttagctgtt tctccaggcc
     1381 acatatgatt agttccaggc cgaaaaggaa ggcaggttcg gctccctgcc ggtcgaacag
     1441 ctcaattgct tgtttcagaa gtgggggcat agaatcggtg gtaggtgtct ctctttcctc
     1501 ttttgctact tgatgctcct gttcctccaa tacgcagccc agtgtaaagt ggcccacggc
     1561 ggacagagcg tacagtgcgt tctccaggga gaagccttgc tgacacagga acgcgagctg
     1621 attttccagg gtttcgtact gtttctctgt tgggcgggtg ccgagatgca ctttagcccc
     1681 gtcgcgatgt gagaggagag cacagcggta tgacttggcg ttgttccgca gaaagtcttg
     1741 ccatgactcg ccttccaggg ggcaggagtg ggtatgatgc ctgtccagca tctcgattgg
     1801 cagggcatcg agcagggccc gcttgttctt cacgtgccag tacagggtag gctgctcaac
     1861 tcccagcttt tgagcgagtt tccttgtcgt caggccttcg ataccgactc cattgagtaa
     1921 ttccagagca gagtttatga ctttgctctt gtccagtcta gacatagtat aacttcgtat
     1981 aggatacttt atacgaagtt atcattggga ttcttcctat tttgatccaa gcatcaccat
     2041 cgaccctcta gtccagatct caccatcgac ccataacttc gtatagcata cattatacga
     2101 agttataacc cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg
     2161 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata
     2221 aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt
     2281 ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt
     2341 gggctctatg gcttctgagg cggaaagaac cagatcctct cttaaggtag catcgagatt
     2401 taaattaggg ataacagggt aatggcgcgg gccgcaggaa cccctagtga tggagttggc
     2461 cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg
     2521 cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg
     2581 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag
     2641 caaccatagt acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     2701 agcgtgaccg ctacacttgc cagcgcctta gcgcccgctc ctttcgcttt cttcccttcc
     2761 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     2821 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgatttggg tgatggttca
     2881 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     2941 tttaatagtg gactcttgtt ccaaactgga acaacactca actctatctc gggctattct
     3001 tttgatttat aagggatttt gccgatttcg gtctattggt taaaaaatga gctgatttaa
     3061 caaaaattta acgcgaattt taacaaaata ttaacgttta caattttatg gtgcactctc
     3121 agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct
     3181 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     3241 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag
     3301 ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg
     3361 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     3421 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     3481 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     3541 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     3601 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     3661 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     3721 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     3781 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     3841 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     3901 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     3961 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     4021 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     4081 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     4141 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     4201 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     4261 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     4321 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     4381 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     4441 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     4501 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     4561 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     4621 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
     4681 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     4741 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     4801 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     4861 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     4921 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     4981 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     5041 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     5101 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     5161 tttgctcaca tgt
//
