LOCUS       sequence-365492-        7072 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  One in a series of plasmids to titrate levels of expressed protein 
            to levels of endogenous protein (attenuated Tet-On promoter, Fig 3).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7072)
  AUTHORS   Millette MM, Holland ED, Tenpas TJ, Dent EW
  TITLE     A Single Transcript Knockdown-Replacement Strategy Employing 5' UTR 
            Secondary Structures to Precisely Titrate Rescue Protein 
            Translation.
  JOURNAL   Front Genome Ed. 2022 Mar 28;4:803375. doi: 
            10.3389/fgeed.2022.803375. eCollection 2022.
  PUBMED    35419562
REFERENCE   2  (bases 1 to 7072)
  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: "Front 
            Genome Ed."; date: "2022-03-28"; volume: "4:803375. doi"; pages: " 
            10.3389/fgeed.2022.803375. eCollection 2022"
COMMENT     Sequence Label: pKDR-TetOn.4
FEATURES             Location/Qualifiers
     source          1..7072
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        56..370
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    64..82
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    100..118
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    135..153
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    171..189
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    207..225
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    242..260
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    278..296
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     343..367
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      413..422
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(419..421,422..424,425..1138)
                     /codon_start=1
                     /product="bright green-yellow fluorescent protein derived 
                     from mClover2, with improved photostability (Bajar et al., 
                     2003)"
                     /label=mClover3
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHYVYITADKQKNCIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSKLSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(464..485)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     1072..1093
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    join(1169..1743,1744..1746,1747..1755)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1336..1353)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1563..1582
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     ncRNA           2679..2693
                     /label=miR-30a loop
                     /note="loop from the the 71-nt precursor of the human 
                     miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     promoter        2871..3381
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     3300..3318
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             join(3391..4008,4009..4014,4015..4050,4051..4053,4054..4089,
                     4090..4092,4093..4128,4129..4137)
                     /codon_start=1
                     /product="improved tetracycline-controlled transactivator"
                     /label=rtTA-Advanced
                     /note="In the Tet-On(R) system, rtTA-Advanced binds to the 
                     Tet-responsive element and stimulates transcription only in
                     the presence of tetracycline or doxycycline."
                     /translation="MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     polyA_signal    4185..4240
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4239..4258)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4601..4617)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4601..4617)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4614..4636)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4625..4641
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(4649..4655,4656..4673,4674..4679))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4694..4715
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4774..4969
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4820..4955
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4975..5109
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5012..5031)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5066..5085
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5177..5194)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5348..5936)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5428..5447)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6107..6898,6899..6967))
                     /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     6730..6749
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6968..7072)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cacatttccc cgaaaagtgc cacctggtcg tcgactggcc gcgaatcgat atgtcgagtt
       61 tactccctat cagtgataga gaacgtatgt cgagtttact ccctatcagt gatagagaac
      121 gatgtcgagt ttactcccta tcagtgatag agaacgtatg tcgagtttac tccctatcag
      181 tgatagagaa cgtatgtcga gtttactccc tatcagtgat agagaacgta tgtcgagttt
      241 atccctatca gtgatagaga acgtatgtcg agtttactcc ctatcagtga tagagaacgt
      301 atgtcgaggt aggcgtgtac ggtgggaggc ctatataagc agagctcgtt tagtgaaccg
      361 tcagatcgcc tggagaaacc ggtcggccgc agccctatac tgggctgcgg ccgccaccat
      421 ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg
      481 cgacgtaaac ggccacaagt tcagcgtccg cggcgagggc gagggcgatg ccaccaacgg
      541 caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct
      601 cgtgaccacc ttcggctacg gcgtggcctg cttcagccgc taccccgacc acatgaagca
      661 gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatctcttt
      721 caaggacgac ggtacctaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt
      781 gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa
      841 gctggagtac aacttcaaca gccactacgt ctatatcacg gccgacaagc agaagaactg
      901 catcaaggct aacttcaaga tccgccacaa cgttgaggac ggcagcgtgc agctcgccga
      961 ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta
     1021 cctgagccat cagtccaagc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct
     1081 gctggagttc gtgaccgccg ccgggattac acatggcatg gacgagctgt acaagtagcc
     1141 tgcagggtca cctaaataat tccgcccccc cctctccctc ccccccccct aacgttactg
     1201 gccgaagccg cttggaataa ggccggtgtg cgtttgtcta tatgttattt tccaccatat
     1261 tgccgtcttt tggcaatgtg agggcccgga aacctggccc tgtcttcttg acgagcattc
     1321 ctaggggtct ttcccctctc gccaaaggaa tgcaaggtct gttgaatgtc gtgaaggaag
     1381 cagttcctct ggaagcttct tgaagacaaa caacgtctgt agcgaccctt tgcaggcagc
     1441 ggaacccccc acctggcgac aggtgcctct gcggccaaaa gccacgtgta taagatacac
     1501 ctgcaaaggc ggcacaaccc cagtgccacg ttgtgagttg gatagttgtg gaaagagtca
     1561 aatggctctc ctcaagcgta ttcaacaagg ggctgaagga tgcccagaag gtaccccatt
     1621 gtatgggatc tgatctgggg cctcggtgca catgctttac atgtgtttag tcgaggttaa
     1681 aaaaacgtct aggccccccg aaccacgggg acgtggtttt cctttgaaaa acacgatgat
     1741 aatatggcca caaccatggg ctgcaccgtg agcgccgagg acaaggcggc ggccgagcgc
     1801 ggctctggtt ccggcactag tgtgagtaaa ggagaggccg ttataaaaga gttcatgcgg
     1861 ttcaaggtac acatggaggg atcaatgaac ggacacgagt tcgaaatcga aggggaagga
     1921 gagggcaggc cctatgaggg tactcagacc gccaaactca aagtgacaaa gggaggccct
     1981 ttgcctttct cttgggacat cttgtcacca caatttatgt atggctcccg agcatttata
     2041 aaacacccag cagacatacc cgattactac aagcagtctt tcccagaagg ttttaaatgg
     2101 gaaagggtca tgaactttga ggatggtggt gctgtgacgg tgacgcagga tacttctttg
     2161 gaggacggaa cactcatcta caaggtaaag ctcaggggca cgaacttccc gccggacgga
     2221 ccagtcatgc agaaaaagac tatgggatgg gaggcttcca ccgaacggtt gtatcccgag
     2281 gacggtgttt tgaagggaga tataaaaatg gctcttcggc tgaaagatgg aggcagatac
     2341 ttggcagatt ttaagaccac gtataaggca aaaaaaccag tgcaaatgcc gggagcatat
     2401 aacgttgacc gcaaactgga catcacctca cataacgagg attatacagt agtagaacag
     2461 tacgaaagaa gcgaagggag gcacagcacc gggggtatgg atgagctgta taagtgatag
     2521 gcatgctgtt tgaatgaggc ttcagtactt tacagaatcg ttgcctgcac atcttggaaa
     2581 cacttgctgg gattacttcg acttcttaac ccaacagaag gctcgagaag gtatattgct
     2641 gttgacagtg agcgcactct ttctaggagg ttgtgatagt gaagccacag atgtatcaca
     2701 acctcctaga aagagtatgc ctactgcctc ggacttcaag gggctagaat tcgagcaatt
     2761 atcttgttta ctaaaactga ataccttgct atctctttga tacattttta caaagctgaa
     2821 ttaaaatggt ataaattaaa tcactttttt caattgacgc gtcatgctcc ggggttgggg
     2881 ttgcgccttt tccaaggcag ccctgggttt gcgcagggac gcggctgctc tgggcgtggt
     2941 tccgggaaac gcagcggcgc cgaccctggg tctcgcacat tcttcacgtc cgttcgcagc
     3001 gtcacccgga tcttcgccgc tacccttgtg ggccccccgg cgacgcttcc tgctccgccc
     3061 ctaagtcggg aaggttcctt gcggttcgcg gcgtgccgga cgtgacaaac ggaagccgca
     3121 cgtctcacta gtaccctcgc agacggacag cgccagggag caatggcagc gcgccgaccg
     3181 cgatgggctg tggccaatag cggctgctca gcagggcgcg ccgagagcag cggccgggaa
     3241 ggggcggtgc gggaggcggg gtgtggggcg gtagtgtggg ccctgttcct gcccgcgcgg
     3301 tgttccgcat tctgcaagcc tccggagcgc acgtcggcag tcggctccct cgttgaccga
     3361 atcaccgacc tctctcccca gcaattcacc atgtctagac tggacaagag caaagtcata
     3421 aacggagctc tggaattact caatggtgtc ggtatcgaag gcctgacgac aaggaaactc
     3481 gctcaaaagc tgggagttga gcagcctacc ctgtactggc acgtgaagaa caagcgggcc
     3541 ctgctcgatg ccctgccaat cgagatgctg gacaggcatc atacccactt ctgccccctg
     3601 gaaggcgagt catggcaaga ctttctgcgg aacaacgcca agtcataccg ctgtgctctc
     3661 ctctcacatc gcgacggggc taaagtgcat ctcggcaccc gcccaacaga gaaacagtac
     3721 gaaaccctgg aaaatcagct cgcgttcctg tgtcagcaag gcttctccct ggagaacgca
     3781 ctgtacgctc tgtccgccgt gggccacttt acactgggct gcgtattgga ggaacaggag
     3841 catcaagtag caaaagagga aagagagaca cctaccaccg attctatgcc cccacttctg
     3901 agacaagcaa ttgagctgtt cgaccggcag ggagccgaac ctgccttcct tttcggcctg
     3961 gaactaatca tatgtggcct ggagaaacag ctaaagtgcg aaagcggcgg gccgaccgac
     4021 gcccttgacg attttgactt agacatgctc ccagccgatg cccttgacga ctttgacctt
     4081 gatatgctgc ctgctgacgc tcttgacgat tttgaccttg acatgctccc cgggtaacta
     4141 agtaaggatc gatccaaggt accttgagca tctgacttct ggctaataaa ggaaatttat
     4201 tttcattgca atagtgtgtt ggaatttttt gtgtctctca ctcggaagga catatgggag
     4261 ggcaaatcat ttaaaacatc agaatgagta tttggtttag agtttggcaa catatgccca
     4321 tatgctggct gccatgaaca aaggttggct ataaagaggt catcagtata tgaaacagcc
     4381 ccctgctgtc cattccttat tccatagaaa agccttgact tgaggttaga ttttttttat
     4441 attttgtttt gtgttatttt tttctttaac atccctaaaa ttttccttac atgttttact
     4501 agccagattt ttcctcctct cctgactact cccagtcata gctgtccctc ttctcttatg
     4561 gagatccctc gacctgcagc ccaagcttgg cgtaatcatg gtcatagctg tttcctgtgt
     4621 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
     4681 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
     4741 tccagtcggg aaacctgtcg tgccagcgga tccgcatctc aattagtcag caaccatagt
     4801 cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc
     4861 ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct
     4921 attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctaacttg
     4981 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     5041 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat
     5101 gtctggatcc gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     5161 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     5221 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     5281 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     5341 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     5401 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     5461 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     5521 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     5581 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     5641 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     5701 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     5761 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     5821 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     5881 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     5941 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     6001 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     6061 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     6121 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     6181 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     6241 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     6301 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     6361 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     6421 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     6481 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     6541 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     6601 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     6661 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     6721 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     6781 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     6841 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     6901 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     6961 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     7021 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cg
//
