LOCUS       sequence-365382-        7076 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 7076)
  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 7076)
  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.13
FEATURES             Location/Qualifiers
     source          1..7076
                     /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      417..426
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(423..425,426..428,429..1142)
                     /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(468..489)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     1076..1097
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    join(1173..1747,1748..1750,1751..1759)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1340..1357)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1567..1586
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     ncRNA           2683..2697
                     /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        2875..3385
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     3304..3322
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             join(3395..4012,4013..4018,4019..4054,4055..4057,4058..4093,
                     4094..4096,4097..4132,4133..4141)
                     /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    4189..4244
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4243..4262)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4605..4621)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4605..4621)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4618..4640)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4629..4645
                     /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(4653..4659,4660..4677,4678..4683))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4698..4719
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4778..4973
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4824..4959
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4979..5113
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5016..5035)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5070..5089
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5181..5198)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5352..5940)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5432..5451)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6111..6902,6903..6971))
                     /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     6734..6753
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6972..7076)
                     /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 gcacgcctat actggcgtgc gcggccgcca
      421 ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg
      481 acggcgacgt aaacggccac aagttcagcg tccgcggcga gggcgagggc gatgccacca
      541 acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca
      601 ccctcgtgac caccttcggc tacggcgtgg cctgcttcag ccgctacccc gaccacatga
      661 agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct
      721 ctttcaagga cgacggtacc tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc
      781 tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc
      841 acaagctgga gtacaacttc aacagccact acgtctatat cacggccgac aagcagaaga
      901 actgcatcaa ggctaacttc aagatccgcc acaacgttga ggacggcagc gtgcagctcg
      961 ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc
     1021 actacctgag ccatcagtcc aagctgagca aagaccccaa cgagaagcgc gatcacatgg
     1081 tcctgctgga gttcgtgacc gccgccggga ttacacatgg catggacgag ctgtacaagt
     1141 agcctgcagg gtcacctaaa taattccgcc cccccctctc cctccccccc ccctaacgtt
     1201 actggccgaa gccgcttgga ataaggccgg tgtgcgtttg tctatatgtt attttccacc
     1261 atattgccgt cttttggcaa tgtgagggcc cggaaacctg gccctgtctt cttgacgagc
     1321 attcctaggg gtctttcccc tctcgccaaa ggaatgcaag gtctgttgaa tgtcgtgaag
     1381 gaagcagttc ctctggaagc ttcttgaaga caaacaacgt ctgtagcgac cctttgcagg
     1441 cagcggaacc ccccacctgg cgacaggtgc ctctgcggcc aaaagccacg tgtataagat
     1501 acacctgcaa aggcggcaca accccagtgc cacgttgtga gttggatagt tgtggaaaga
     1561 gtcaaatggc tctcctcaag cgtattcaac aaggggctga aggatgccca gaaggtaccc
     1621 cattgtatgg gatctgatct ggggcctcgg tgcacatgct ttacatgtgt ttagtcgagg
     1681 ttaaaaaaac gtctaggccc cccgaaccac ggggacgtgg ttttcctttg aaaaacacga
     1741 tgataatatg gccacaacca tgggctgcac cgtgagcgcc gaggacaagg cggcggccga
     1801 gcgcggctct ggttccggca ctagtgtgag taaaggagag gccgttataa aagagttcat
     1861 gcggttcaag gtacacatgg agggatcaat gaacggacac gagttcgaaa tcgaagggga
     1921 aggagagggc aggccctatg agggtactca gaccgccaaa ctcaaagtga caaagggagg
     1981 ccctttgcct ttctcttggg acatcttgtc accacaattt atgtatggct cccgagcatt
     2041 tataaaacac ccagcagaca tacccgatta ctacaagcag tctttcccag aaggttttaa
     2101 atgggaaagg gtcatgaact ttgaggatgg tggtgctgtg acggtgacgc aggatacttc
     2161 tttggaggac ggaacactca tctacaaggt aaagctcagg ggcacgaact tcccgccgga
     2221 cggaccagtc atgcagaaaa agactatggg atgggaggct tccaccgaac ggttgtatcc
     2281 cgaggacggt gttttgaagg gagatataaa aatggctctt cggctgaaag atggaggcag
     2341 atacttggca gattttaaga ccacgtataa ggcaaaaaaa ccagtgcaaa tgccgggagc
     2401 atataacgtt gaccgcaaac tggacatcac ctcacataac gaggattata cagtagtaga
     2461 acagtacgaa agaagcgaag ggaggcacag caccgggggt atggatgagc tgtataagtg
     2521 ataggcatgc tgtttgaatg aggcttcagt actttacaga atcgttgcct gcacatcttg
     2581 gaaacacttg ctgggattac ttcgacttct taacccaaca gaaggctcga gaaggtatat
     2641 tgctgttgac agtgagcgca ctctttctag gaggttgtga tagtgaagcc acagatgtat
     2701 cacaacctcc tagaaagagt atgcctactg cctcggactt caaggggcta gaattcgagc
     2761 aattatcttg tttactaaaa ctgaatacct tgctatctct ttgatacatt tttacaaagc
     2821 tgaattaaaa tggtataaat taaatcactt ttttcaattg acgcgtcatg ctccggggtt
     2881 ggggttgcgc cttttccaag gcagccctgg gtttgcgcag ggacgcggct gctctgggcg
     2941 tggttccggg aaacgcagcg gcgccgaccc tgggtctcgc acattcttca cgtccgttcg
     3001 cagcgtcacc cggatcttcg ccgctaccct tgtgggcccc ccggcgacgc ttcctgctcc
     3061 gcccctaagt cgggaaggtt ccttgcggtt cgcggcgtgc cggacgtgac aaacggaagc
     3121 cgcacgtctc actagtaccc tcgcagacgg acagcgccag ggagcaatgg cagcgcgccg
     3181 accgcgatgg gctgtggcca atagcggctg ctcagcaggg cgcgccgaga gcagcggccg
     3241 ggaaggggcg gtgcgggagg cggggtgtgg ggcggtagtg tgggccctgt tcctgcccgc
     3301 gcggtgttcc gcattctgca agcctccgga gcgcacgtcg gcagtcggct ccctcgttga
     3361 ccgaatcacc gacctctctc cccagcaatt caccatgtct agactggaca agagcaaagt
     3421 cataaacgga gctctggaat tactcaatgg tgtcggtatc gaaggcctga cgacaaggaa
     3481 actcgctcaa aagctgggag ttgagcagcc taccctgtac tggcacgtga agaacaagcg
     3541 ggccctgctc gatgccctgc caatcgagat gctggacagg catcataccc acttctgccc
     3601 cctggaaggc gagtcatggc aagactttct gcggaacaac gccaagtcat accgctgtgc
     3661 tctcctctca catcgcgacg gggctaaagt gcatctcggc acccgcccaa cagagaaaca
     3721 gtacgaaacc ctggaaaatc agctcgcgtt cctgtgtcag caaggcttct ccctggagaa
     3781 cgcactgtac gctctgtccg ccgtgggcca ctttacactg ggctgcgtat tggaggaaca
     3841 ggagcatcaa gtagcaaaag aggaaagaga gacacctacc accgattcta tgcccccact
     3901 tctgagacaa gcaattgagc tgttcgaccg gcagggagcc gaacctgcct tccttttcgg
     3961 cctggaacta atcatatgtg gcctggagaa acagctaaag tgcgaaagcg gcgggccgac
     4021 cgacgccctt gacgattttg acttagacat gctcccagcc gatgcccttg acgactttga
     4081 ccttgatatg ctgcctgctg acgctcttga cgattttgac cttgacatgc tccccgggta
     4141 actaagtaag gatcgatcca aggtaccttg agcatctgac ttctggctaa taaaggaaat
     4201 ttattttcat tgcaatagtg tgttggaatt ttttgtgtct ctcactcgga aggacatatg
     4261 ggagggcaaa tcatttaaaa catcagaatg agtatttggt ttagagtttg gcaacatatg
     4321 cccatatgct ggctgccatg aacaaaggtt ggctataaag aggtcatcag tatatgaaac
     4381 agccccctgc tgtccattcc ttattccata gaaaagcctt gacttgaggt tagatttttt
     4441 ttatattttg ttttgtgtta tttttttctt taacatccct aaaattttcc ttacatgttt
     4501 tactagccag atttttcctc ctctcctgac tactcccagt catagctgtc cctcttctct
     4561 tatggagatc cctcgacctg cagcccaagc ttggcgtaat catggtcata gctgtttcct
     4621 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     4681 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     4741 gctttccagt cgggaaacct gtcgtgccag cggatccgca tctcaattag tcagcaacca
     4801 tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc
     4861 cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg
     4921 agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagctaa
     4981 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     5041 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     5101 tcatgtctgg atccgctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     5161 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     5221 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     5281 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     5341 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     5401 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     5461 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     5521 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     5581 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     5641 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     5701 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     5761 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     5821 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     5881 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     5941 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     6001 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     6061 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     6121 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     6181 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     6241 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     6301 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     6361 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     6421 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     6481 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     6541 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     6601 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     6661 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     6721 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     6781 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     6841 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     6901 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     6961 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     7021 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcg
//
