LOCUS       sequence-365363-        7055 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 (non-attenuated Tet-On promoter).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7055)
  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 7055)
  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
FEATURES             Location/Qualifiers
     source          1..7055
                     /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      396..405
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(402..404,405..407,408..1121)
                     /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(447..468)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     1055..1076
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    join(1152..1726,1727..1729,1730..1738)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1319..1336)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1546..1565
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     ncRNA           2662..2676
                     /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        2854..3364
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     3283..3301
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             join(3374..3991,3992..3997,3998..4033,4034..4036,4037..4072,
                     4073..4075,4076..4111,4112..4120)
                     /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    4168..4223
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4222..4241)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4584..4600)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4584..4600)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4597..4619)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4608..4624
                     /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(4632..4638,4639..4656,4657..4662))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4677..4698
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4757..4952
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4803..4938
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4958..5092
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4995..5014)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5049..5068
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5160..5177)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5331..5919)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5411..5430)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6090..6881,6882..6950))
                     /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     6713..6732
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6951..7055)
                     /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 ggttatactg cggccgccac catggtgagc aagggcgagg
      421 agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca
      481 agttcagcgt ccgcggcgag ggcgagggcg atgccaccaa cggcaagctg accctgaagt
      541 tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accttcggct
      601 acggcgtggc ctgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt
      661 ccgccatgcc cgaaggctac gtccaggagc gcaccatctc tttcaaggac gacggtacct
      721 acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga
      781 agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag tacaacttca
      841 acagccacta cgtctatatc acggccgaca agcagaagaa ctgcatcaag gctaacttca
      901 agatccgcca caacgttgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca
      961 cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc catcagtcca
     1021 agctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg
     1081 ccgccgggat tacacatggc atggacgagc tgtacaagta gcctgcaggg tcacctaaat
     1141 aattccgccc ccccctctcc ctcccccccc cctaacgtta ctggccgaag ccgcttggaa
     1201 taaggccggt gtgcgtttgt ctatatgtta ttttccacca tattgccgtc ttttggcaat
     1261 gtgagggccc ggaaacctgg ccctgtcttc ttgacgagca ttcctagggg tctttcccct
     1321 ctcgccaaag gaatgcaagg tctgttgaat gtcgtgaagg aagcagttcc tctggaagct
     1381 tcttgaagac aaacaacgtc tgtagcgacc ctttgcaggc agcggaaccc cccacctggc
     1441 gacaggtgcc tctgcggcca aaagccacgt gtataagata cacctgcaaa ggcggcacaa
     1501 ccccagtgcc acgttgtgag ttggatagtt gtggaaagag tcaaatggct ctcctcaagc
     1561 gtattcaaca aggggctgaa ggatgcccag aaggtacccc attgtatggg atctgatctg
     1621 gggcctcggt gcacatgctt tacatgtgtt tagtcgaggt taaaaaaacg tctaggcccc
     1681 ccgaaccacg gggacgtggt tttcctttga aaaacacgat gataatatgg ccacaaccat
     1741 gggctgcacc gtgagcgccg aggacaaggc ggcggccgag cgcggctctg gttccggcac
     1801 tagtgtgagt aaaggagagg ccgttataaa agagttcatg cggttcaagg tacacatgga
     1861 gggatcaatg aacggacacg agttcgaaat cgaaggggaa ggagagggca ggccctatga
     1921 gggtactcag accgccaaac tcaaagtgac aaagggaggc cctttgcctt tctcttggga
     1981 catcttgtca ccacaattta tgtatggctc ccgagcattt ataaaacacc cagcagacat
     2041 acccgattac tacaagcagt ctttcccaga aggttttaaa tgggaaaggg tcatgaactt
     2101 tgaggatggt ggtgctgtga cggtgacgca ggatacttct ttggaggacg gaacactcat
     2161 ctacaaggta aagctcaggg gcacgaactt cccgccggac ggaccagtca tgcagaaaaa
     2221 gactatggga tgggaggctt ccaccgaacg gttgtatccc gaggacggtg ttttgaaggg
     2281 agatataaaa atggctcttc ggctgaaaga tggaggcaga tacttggcag attttaagac
     2341 cacgtataag gcaaaaaaac cagtgcaaat gccgggagca tataacgttg accgcaaact
     2401 ggacatcacc tcacataacg aggattatac agtagtagaa cagtacgaaa gaagcgaagg
     2461 gaggcacagc accgggggta tggatgagct gtataagtga taggcatgct gtttgaatga
     2521 ggcttcagta ctttacagaa tcgttgcctg cacatcttgg aaacacttgc tgggattact
     2581 tcgacttctt aacccaacag aaggctcgag aaggtatatt gctgttgaca gtgagcgcac
     2641 tctttctagg aggttgtgat agtgaagcca cagatgtatc acaacctcct agaaagagta
     2701 tgcctactgc ctcggacttc aaggggctag aattcgagca attatcttgt ttactaaaac
     2761 tgaatacctt gctatctctt tgatacattt ttacaaagct gaattaaaat ggtataaatt
     2821 aaatcacttt tttcaattga cgcgtcatgc tccggggttg gggttgcgcc ttttccaagg
     2881 cagccctggg tttgcgcagg gacgcggctg ctctgggcgt ggttccggga aacgcagcgg
     2941 cgccgaccct gggtctcgca cattcttcac gtccgttcgc agcgtcaccc ggatcttcgc
     3001 cgctaccctt gtgggccccc cggcgacgct tcctgctccg cccctaagtc gggaaggttc
     3061 cttgcggttc gcggcgtgcc ggacgtgaca aacggaagcc gcacgtctca ctagtaccct
     3121 cgcagacgga cagcgccagg gagcaatggc agcgcgccga ccgcgatggg ctgtggccaa
     3181 tagcggctgc tcagcagggc gcgccgagag cagcggccgg gaaggggcgg tgcgggaggc
     3241 ggggtgtggg gcggtagtgt gggccctgtt cctgcccgcg cggtgttccg cattctgcaa
     3301 gcctccggag cgcacgtcgg cagtcggctc cctcgttgac cgaatcaccg acctctctcc
     3361 ccagcaattc accatgtcta gactggacaa gagcaaagtc ataaacggag ctctggaatt
     3421 actcaatggt gtcggtatcg aaggcctgac gacaaggaaa ctcgctcaaa agctgggagt
     3481 tgagcagcct accctgtact ggcacgtgaa gaacaagcgg gccctgctcg atgccctgcc
     3541 aatcgagatg ctggacaggc atcataccca cttctgcccc ctggaaggcg agtcatggca
     3601 agactttctg cggaacaacg ccaagtcata ccgctgtgct ctcctctcac atcgcgacgg
     3661 ggctaaagtg catctcggca cccgcccaac agagaaacag tacgaaaccc tggaaaatca
     3721 gctcgcgttc ctgtgtcagc aaggcttctc cctggagaac gcactgtacg ctctgtccgc
     3781 cgtgggccac tttacactgg gctgcgtatt ggaggaacag gagcatcaag tagcaaaaga
     3841 ggaaagagag acacctacca ccgattctat gcccccactt ctgagacaag caattgagct
     3901 gttcgaccgg cagggagccg aacctgcctt ccttttcggc ctggaactaa tcatatgtgg
     3961 cctggagaaa cagctaaagt gcgaaagcgg cgggccgacc gacgcccttg acgattttga
     4021 cttagacatg ctcccagccg atgcccttga cgactttgac cttgatatgc tgcctgctga
     4081 cgctcttgac gattttgacc ttgacatgct ccccgggtaa ctaagtaagg atcgatccaa
     4141 ggtaccttga gcatctgact tctggctaat aaaggaaatt tattttcatt gcaatagtgt
     4201 gttggaattt tttgtgtctc tcactcggaa ggacatatgg gagggcaaat catttaaaac
     4261 atcagaatga gtatttggtt tagagtttgg caacatatgc ccatatgctg gctgccatga
     4321 acaaaggttg gctataaaga ggtcatcagt atatgaaaca gccccctgct gtccattcct
     4381 tattccatag aaaagccttg acttgaggtt agattttttt tatattttgt tttgtgttat
     4441 ttttttcttt aacatcccta aaattttcct tacatgtttt actagccaga tttttcctcc
     4501 tctcctgact actcccagtc atagctgtcc ctcttctctt atggagatcc ctcgacctgc
     4561 agcccaagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     4621 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     4681 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     4741 tcgtgccagc ggatccgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc
     4801 ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt
     4861 tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag
     4921 gaggcttttt tggaggccta ggcttttgca aaaagctaac ttgtttattg cagcttataa
     4981 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
     5041 ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgga tccgctgcat
     5101 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     5161 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     5221 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     5281 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     5341 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     5401 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     5461 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     5521 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     5581 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     5641 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     5701 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     5761 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     5821 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     5881 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     5941 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     6001 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     6061 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     6121 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     6181 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     6241 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     6301 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     6361 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     6421 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     6481 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     6541 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     6601 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     6661 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     6721 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     6781 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     6841 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     6901 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     6961 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     7021 tgtatttaga aaaataaaca aataggggtt ccgcg
//
