LOCUS       sequence-365494-        7074 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 7074)
  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 7074)
  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.9
FEATURES             Location/Qualifiers
     source          1..7074
                     /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      415..424
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(421..423,424..426,427..1140)
                     /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(466..487)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     1074..1095
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    join(1171..1745,1746..1748,1749..1757)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1338..1355)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1565..1584
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     ncRNA           2681..2695
                     /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        2873..3383
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     3302..3320
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             join(3393..4010,4011..4016,4017..4052,4053..4055,4056..4091,
                     4092..4094,4095..4130,4131..4139)
                     /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    4187..4242
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4241..4260)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4603..4619)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4603..4619)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4616..4638)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4627..4643
                     /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(4651..4657,4658..4675,4676..4681))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4696..4717
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4776..4971
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4822..4957
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4977..5111
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5014..5033)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5068..5087
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5179..5196)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5350..5938)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5430..5449)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6109..6900,6901..6969))
                     /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     6732..6751
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6970..7074)
                     /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 ctcggctata ctgccgaggc ggccgccacc
      421 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
      481 ggcgacgtaa acggccacaa gttcagcgtc cgcggcgagg gcgagggcga tgccaccaac
      541 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
      601 ctcgtgacca ccttcggcta cggcgtggcc tgcttcagcc gctaccccga ccacatgaag
      661 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatctct
      721 ttcaaggacg acggtaccta caagacccgc gccgaggtga agttcgaggg cgacaccctg
      781 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
      841 aagctggagt acaacttcaa cagccactac gtctatatca cggccgacaa gcagaagaac
      901 tgcatcaagg ctaacttcaa gatccgccac aacgttgagg acggcagcgt gcagctcgcc
      961 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
     1021 tacctgagcc atcagtccaa gctgagcaaa gaccccaacg agaagcgcga tcacatggtc
     1081 ctgctggagt tcgtgaccgc cgccgggatt acacatggca tggacgagct gtacaagtag
     1141 cctgcagggt cacctaaata attccgcccc cccctctccc tccccccccc ctaacgttac
     1201 tggccgaagc cgcttggaat aaggccggtg tgcgtttgtc tatatgttat tttccaccat
     1261 attgccgtct tttggcaatg tgagggcccg gaaacctggc cctgtcttct tgacgagcat
     1321 tcctaggggt ctttcccctc tcgccaaagg aatgcaaggt ctgttgaatg tcgtgaagga
     1381 agcagttcct ctggaagctt cttgaagaca aacaacgtct gtagcgaccc tttgcaggca
     1441 gcggaacccc ccacctggcg acaggtgcct ctgcggccaa aagccacgtg tataagatac
     1501 acctgcaaag gcggcacaac cccagtgcca cgttgtgagt tggatagttg tggaaagagt
     1561 caaatggctc tcctcaagcg tattcaacaa ggggctgaag gatgcccaga aggtacccca
     1621 ttgtatggga tctgatctgg ggcctcggtg cacatgcttt acatgtgttt agtcgaggtt
     1681 aaaaaaacgt ctaggccccc cgaaccacgg ggacgtggtt ttcctttgaa aaacacgatg
     1741 ataatatggc cacaaccatg ggctgcaccg tgagcgccga ggacaaggcg gcggccgagc
     1801 gcggctctgg ttccggcact agtgtgagta aaggagaggc cgttataaaa gagttcatgc
     1861 ggttcaaggt acacatggag ggatcaatga acggacacga gttcgaaatc gaaggggaag
     1921 gagagggcag gccctatgag ggtactcaga ccgccaaact caaagtgaca aagggaggcc
     1981 ctttgccttt ctcttgggac atcttgtcac cacaatttat gtatggctcc cgagcattta
     2041 taaaacaccc agcagacata cccgattact acaagcagtc tttcccagaa ggttttaaat
     2101 gggaaagggt catgaacttt gaggatggtg gtgctgtgac ggtgacgcag gatacttctt
     2161 tggaggacgg aacactcatc tacaaggtaa agctcagggg cacgaacttc ccgccggacg
     2221 gaccagtcat gcagaaaaag actatgggat gggaggcttc caccgaacgg ttgtatcccg
     2281 aggacggtgt tttgaaggga gatataaaaa tggctcttcg gctgaaagat ggaggcagat
     2341 acttggcaga ttttaagacc acgtataagg caaaaaaacc agtgcaaatg ccgggagcat
     2401 ataacgttga ccgcaaactg gacatcacct cacataacga ggattataca gtagtagaac
     2461 agtacgaaag aagcgaaggg aggcacagca ccgggggtat ggatgagctg tataagtgat
     2521 aggcatgctg tttgaatgag gcttcagtac tttacagaat cgttgcctgc acatcttgga
     2581 aacacttgct gggattactt cgacttctta acccaacaga aggctcgaga aggtatattg
     2641 ctgttgacag tgagcgcact ctttctagga ggttgtgata gtgaagccac agatgtatca
     2701 caacctccta gaaagagtat gcctactgcc tcggacttca aggggctaga attcgagcaa
     2761 ttatcttgtt tactaaaact gaataccttg ctatctcttt gatacatttt tacaaagctg
     2821 aattaaaatg gtataaatta aatcactttt ttcaattgac gcgtcatgct ccggggttgg
     2881 ggttgcgcct tttccaaggc agccctgggt ttgcgcaggg acgcggctgc tctgggcgtg
     2941 gttccgggaa acgcagcggc gccgaccctg ggtctcgcac attcttcacg tccgttcgca
     3001 gcgtcacccg gatcttcgcc gctacccttg tgggcccccc ggcgacgctt cctgctccgc
     3061 ccctaagtcg ggaaggttcc ttgcggttcg cggcgtgccg gacgtgacaa acggaagccg
     3121 cacgtctcac tagtaccctc gcagacggac agcgccaggg agcaatggca gcgcgccgac
     3181 cgcgatgggc tgtggccaat agcggctgct cagcagggcg cgccgagagc agcggccggg
     3241 aaggggcggt gcgggaggcg gggtgtgggg cggtagtgtg ggccctgttc ctgcccgcgc
     3301 ggtgttccgc attctgcaag cctccggagc gcacgtcggc agtcggctcc ctcgttgacc
     3361 gaatcaccga cctctctccc cagcaattca ccatgtctag actggacaag agcaaagtca
     3421 taaacggagc tctggaatta ctcaatggtg tcggtatcga aggcctgacg acaaggaaac
     3481 tcgctcaaaa gctgggagtt gagcagccta ccctgtactg gcacgtgaag aacaagcggg
     3541 ccctgctcga tgccctgcca atcgagatgc tggacaggca tcatacccac ttctgccccc
     3601 tggaaggcga gtcatggcaa gactttctgc ggaacaacgc caagtcatac cgctgtgctc
     3661 tcctctcaca tcgcgacggg gctaaagtgc atctcggcac ccgcccaaca gagaaacagt
     3721 acgaaaccct ggaaaatcag ctcgcgttcc tgtgtcagca aggcttctcc ctggagaacg
     3781 cactgtacgc tctgtccgcc gtgggccact ttacactggg ctgcgtattg gaggaacagg
     3841 agcatcaagt agcaaaagag gaaagagaga cacctaccac cgattctatg cccccacttc
     3901 tgagacaagc aattgagctg ttcgaccggc agggagccga acctgccttc cttttcggcc
     3961 tggaactaat catatgtggc ctggagaaac agctaaagtg cgaaagcggc gggccgaccg
     4021 acgcccttga cgattttgac ttagacatgc tcccagccga tgcccttgac gactttgacc
     4081 ttgatatgct gcctgctgac gctcttgacg attttgacct tgacatgctc cccgggtaac
     4141 taagtaagga tcgatccaag gtaccttgag catctgactt ctggctaata aaggaaattt
     4201 attttcattg caatagtgtg ttggaatttt ttgtgtctct cactcggaag gacatatggg
     4261 agggcaaatc atttaaaaca tcagaatgag tatttggttt agagtttggc aacatatgcc
     4321 catatgctgg ctgccatgaa caaaggttgg ctataaagag gtcatcagta tatgaaacag
     4381 ccccctgctg tccattcctt attccataga aaagccttga cttgaggtta gatttttttt
     4441 atattttgtt ttgtgttatt tttttcttta acatccctaa aattttcctt acatgtttta
     4501 ctagccagat ttttcctcct ctcctgacta ctcccagtca tagctgtccc tcttctctta
     4561 tggagatccc tcgacctgca gcccaagctt ggcgtaatca tggtcatagc tgtttcctgt
     4621 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     4681 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     4741 tttccagtcg ggaaacctgt cgtgccagcg gatccgcatc tcaattagtc agcaaccata
     4801 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg
     4861 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag
     4921 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctaact
     4981 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     5041 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
     5101 atgtctggat ccgctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     5161 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     5221 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     5281 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     5341 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     5401 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     5461 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     5521 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     5581 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     5641 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     5701 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     5761 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     5821 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     5881 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     5941 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     6001 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     6061 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     6121 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     6181 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     6241 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     6301 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     6361 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     6421 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     6481 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     6541 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     6601 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     6661 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     6721 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     6781 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     6841 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     6901 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     6961 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     7021 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcg
//
