LOCUS       sequence-7020-ex        7117 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7117)
  AUTHORS   Wong ET, Kolman JL, Li YC, Mesner LD, Hillen W, Berens C, Wahl GM
  TITLE     Reproducible doxycycline-inducible transgene expression at specific 
            loci generated by Cre-recombinase mediated cassette exchange.
  JOURNAL   Nucleic Acids Res. 2005  . 33(17):e147.
  PUBMED    16204450
REFERENCE   2  (bases 1 to 7117)
  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: "Nucleic 
            Acids Res. 2005  . 33(17):e147."
COMMENT     Sequence Label: pL3-TRE-LucGFP-2L
FEATURES             Location/Qualifiers
     source          1..7117
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     428..444
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    477..510
                     /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    join(541..559,560..582,583..601,602..624,625..643,644..666,
                     667..685,686..708,709..727,728..750,751..769,770..792,
                     793..811)
                     /label=tetracycline response element
                     /bound_moiety="tetracycline repressor TetR"
                     /note="contains seven copies of the tetracycline operator 
                     tetO"
     promoter        844..882
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     879..903
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      1040..1049
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1046..2695
                     /codon_start=1
                     /gene="luc+"
                     /product="firefly luciferase"
                     /label=luciferase
                     /note="enhanced luc+ version of the luciferase gene"
                     /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA
                     HIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP
                     ANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS
                     MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
                     RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK
                     IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY
                     GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS
                     GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL
                     LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV
                     FVDEVPKGLTGKLDARKIREILIKAKKGGKIAV"
     primer_bind     complement(1112..1130)
                     /label=LucNrev
                     /note="Firefly luciferase, reverse primer"
     regulatory      2711..2720
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2717..2719,2720..2722,2723..3436)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2717..2719,2720..2722,2723..3433)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2762..2783)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3023..3042)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3370..3391
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     intron          3491..4063
                     /label=beta-globin intron
                     /note="intron from rabbit beta-globin gene"
     primer_bind     complement(4195..4214)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4260..4315
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4314..4333)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     protein_bind    complement(4829..4862)
                     /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(4896..4912)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4896..4912)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4909..4931)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4920..4936
                     /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(4944..4950,4951..4968,4969..4974))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4989..5010
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5127..5144)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5298..5886)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5378..5397)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6057..6848,6849..6917))
                     /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     6680..6699
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6918..7022)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7090..7108
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acccgggggc
      421 gcgccttgta aaacgacggc cagtgaattc tcgatctaga gaaccccttc atcgagataa
      481 cttcgtataa agtctcctat acgaagttat atcgatgaac cccttcctcg agtttaccac
      541 tccctatcag tgatagagaa aagtgaaagt cgagtttacc actccctatc agtgatagag
      601 aaaagtgaaa gtcgagttta ccactcccta tcagtgatag agaaaagtga aagtcgagtt
      661 taccactccc tatcagtgat agagaaaagt gaaagtcgag tttaccactc cctatcagtg
      721 atagagaaaa gtgaaagtcg agtttaccac tccctatcag tgatagagaa aagtgaaagt
      781 cgagtttacc actccctatc agtgatagag aaaagtgaaa gtcgagctcg gtacccgggt
      841 cgaggtaggc gtgtacggtg ggaggcctat ataagcagag ctcgtttagt gaaccgtcag
      901 atcgcctgga gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc
      961 agcctccgcg gccccgaatt cgagctcggt acccggggat ctcgagctct aagcttggca
     1021 ttccggtact gttggtaaag ccaccatgga agacgccaaa aacataaaga aaggcccggc
     1081 gccattctat ccgctggaag atggaaccgc tggagagcaa ctgcataagg ctatgaagag
     1141 atacgccctg gttcctggaa caattgcttt tacagatgca catatcgagg tggacatcac
     1201 ttacgctgag tacttcgaaa tgtccgttcg gttggcagaa gctatgaaac gatatgggct
     1261 gaatacaaat cacagaatcg tcgtatgcag tgaaaactct cttcaattct ttatgccggt
     1321 gttgggcgcg ttatttatcg gagttgcagt tgcgcccgcg aacgacattt ataatgaacg
     1381 tgaattgctc aacagtatgg gcatttcgca gcctaccgtg gtgttcgttt ccaaaaaggg
     1441 gttgcaaaaa attttgaacg tgcaaaaaaa gctcccaatc atccaaaaaa ttattatcat
     1501 ggattctaaa acggattacc agggatttca gtcgatgtac acgttcgtca catctcatct
     1561 acctcccggt tttaatgaat acgattttgt gccagagtcc ttcgataggg acaagacaat
     1621 tgcactgatc atgaactcct ctggatctac tggtctgcct aaaggtgtcg ctctgcctca
     1681 tagaactgcc tgcgtgagat tctcgcatgc cagagatcct atttttggca atcaaatcat
     1741 tccggatact gcgattttaa gtgttgttcc attccatcac ggttttggaa tgtttactac
     1801 actcggatat ttgatatgtg gatttcgagt cgtcttaatg tatagatttg aagaagagct
     1861 gtttctgagg agccttcagg attacaagat tcaaagtgcg ctgctggtgc caaccctatt
     1921 ctccttcttc gccaaaagca ctctgattga caaatacgat ttatctaatt tacacgaaat
     1981 tgcttctggt ggcgctcccc tctctaagga agtcggggaa gcggttgcca agaggttcca
     2041 tctgccaggt atcaggcaag gatatgggct cactgagact acatcagcta ttctgattac
     2101 acccgagggg gatgataaac cgggcgcggt cggtaaagtt gttccatttt ttgaagcgaa
     2161 ggttgtggat ctggataccg ggaaaacgct gggcgttaat caaagaggcg aactgtgtgt
     2221 gagaggtcct atgattatgt ccggttatgt aaacaatccg gaagcgacca acgccttgat
     2281 tgacaaggat ggatggctac attctggaga catagcttac tgggacgaag acgaacactt
     2341 cttcatcgtt gaccgcctga agtctctgat taagtacaaa ggctatcagg tggctcccgc
     2401 tgaattggaa tccatcttgc tccaacaccc caacatcttc gacgcaggtg tcgcaggtct
     2461 tcccgacgat gacgccggtg aacttcccgc cgccgttgtt gttttggagc acggaaagac
     2521 gatgacggaa aaagagatcg tggattacgt cgccagtcaa gtaacaaccg cgaaaaagtt
     2581 gcgcggagga gttgtgtttg tggacgaagt accgaaaggt cttaccggaa aactcgacgc
     2641 aagaaaaatc agagagatcc tcataaaggc caagaagggc ggaaagatcg ccgtgcggga
     2701 tccaccggtc gccaccatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat
     2761 cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga
     2821 gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc
     2881 cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta
     2941 ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca
     3001 ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt
     3061 cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg
     3121 caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc
     3181 cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg
     3241 cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct
     3301 gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa
     3361 gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga
     3421 cgagctgtac aagtaaagcg gccgcatcga taagcttgtc gacgatatct ctagagctga
     3481 gaacttcagg gtgagtttgg ggacccttga ttgttctttc tttttcgcta ttgtaaaatt
     3541 catgttatat ggagggggca aagttttcag ggtgttgttt agaatgggaa gatgtccctt
     3601 gtatcaccat ggaccctcat gataattttg tttctttcac tttctactct gttgacaacc
     3661 attgtctcct cttattttct tttcattttc tgtaactttt tcgttaaact ttagcttgca
     3721 tttgtaacga atttttaaat tcacttttgt ttatttgtca gattgtaagt actttctcta
     3781 atcacttttt tttcaaggca atcagggtat attatattgt acttcagcac agttttagag
     3841 aacaattgtt ataattaaat gataaggtag aatatttctg catataaatt ctggctggcg
     3901 tggaaatatt cttattggta gaaacaacta caccctggtc atcatcctgc ctttctcttt
     3961 atggttacaa tgatatacac tgtttgagat gaggataaaa tactctgagt ccaaaccggg
     4021 cccctctgct aaccatgttc atgccttctt ctctttccta cagctcctgg gcaacgtgct
     4081 ggttgttgtg ctgtctcatc attttggcaa agaattcact cctcaggtgc aggctgccta
     4141 tcagaaggtg gtggctggtg tggccaatgc cctggctcac aaataccact gagatctttt
     4201 tccctctgcc aaaaattatg gggacatcat gaagcccctt gagcatctga cttctggcta
     4261 ataaaggaaa tttattttca ttgcaatagt gtgtgggaat tttttgtgtc tctcactcgg
     4321 aaggacatat gggagggcaa atcatttaaa acatcagaat gagtatttgg tttagagttt
     4381 ggcaacatat gccatatgct ggctgccatg aacaaaggtg gctataaaga ggtcatcagt
     4441 atatgaaaca gccccctgct gtccattcct tattccatag aaaagccttg acttgaggtt
     4501 agattttttt tatattttgt tttgtgttat ttttttcttt aacatcccta aaattttcct
     4561 tacatgtttt actagccaga tttttcctcc tctcctgact actcccagtc atagctgtcc
     4621 ctcttctctt atgaactcga ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     4681 tgcgtattgg gcgctcttcc gctacgtcac cacccccggc tccataccga cgatatgcga
     4741 cctggcgcgc acgtttgccc gggagatggg ggaggctaac tgaagcttcc cgggggtacc
     4801 gaattcgtcg acagatccgg ccggccccat aacttcgtat agcatacatt atacgaagtt
     4861 atcccctgca ggcatgcaag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     4921 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     4981 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     5041 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     5101 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     5161 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     5221 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     5281 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     5341 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     5401 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     5461 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     5521 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     5581 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     5641 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     5701 ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct
     5761 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     5821 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     5881 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     5941 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     6001 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     6061 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     6121 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     6181 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     6241 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     6301 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     6361 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     6421 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     6481 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     6541 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     6601 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     6661 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     6721 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     6781 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     6841 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     6901 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     6961 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     7021 cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta
     7081 acctataaaa ataggcgtat cacgaggccc tttcgtc
//
