LOCUS       sequence-148973-        6059 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  negative control for expression assays.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6059)
  TITLE     pTREtight reporter plasmids--Roderic Guigo
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6059)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pTre-Tight-luciferase-GFP (467)
FEATURES             Location/Qualifiers
     source          1..6059
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        4..318
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    12..30
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    48..66
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    83..101
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    119..137
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    155..173
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    190..208
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    226..244
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     291..315
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     primer_bind     350..369
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        350..368
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(422..424,425..427,428..1138)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKLSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VSFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(467..488)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(728..747)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1075..1096
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_RNA        1283..1301
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        1322..1340
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        1361..1379
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        1400..1418
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        1439..1457
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        1478..1496
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        1517..1535
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        1556..1574
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     primer_bind     complement(1612..1629)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1805..1886
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1897..1914)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2068..2656)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2148..2167)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2827..3618,3619..3687))
                     /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     3450..3469
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3688..3792)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3809..4005
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3856..3991
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     regulatory      4041..4050
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4047..5699
                     /codon_start=1
                     /product="firefly luciferase"
                     /label=luciferase
                     /note="synthetic luc2 version of the luciferase gene"
                     /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA
                     HIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP
                     ANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS
                     MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
                     RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK
                     IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY
                     GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS
                     GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL
                     LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV
                     FVDEVPKGLTGKLDARKIREILIKAKKGGKIAV"
     polyA_signal    5743..5864
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5754..5773)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     6028..6046
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ctcgagttta ctccctatca gtgatagaga acgtatgtcg agtttactcc ctatcagtga
       61 tagagaacga tgtcgagttt actccctatc agtgatagag aacgtatgtc gagtttactc
      121 cctatcagtg atagagaacg tatgtcgagt ttactcccta tcagtgatag agaacgtatg
      181 tcgagtttat ccctatcagt gatagagaac gtatgtcgag tttactccct atcagtgata
      241 gagaacgtat gtcgaggtag gcgtgtacgg tgggaggcct atataagcag agctcgttta
      301 gtgaaccgtc agatcgcctg gagaattcga gctcggtacc cggggatcct aatacgactc
      361 actataggga gacccaagct ggctagcgtt taaacttaag cttggtaccg agctcggatc
      421 catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga
      481 cggcgacgta aacggccaca agctcagcgt gtccggcgag ggcgagggcg atgccaccta
      541 cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac
      601 cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa
      661 gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt
      721 cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct
      781 ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca
      841 caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa
      901 cggcatcaag gtgagcttca agatccgcca caacatcgag gacggcagcg tgcagctcgc
      961 cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca
     1021 ctacctgagc acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt
     1081 cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaaggg
     1141 atccactagt ccagtgtggt ggaattctgc agatatccag cacagtggcg gccgctcgct
     1201 ntcttgctgt ccaatttcta ttaaaggttc ctttgttccc taagtccaac taccaaactg
     1261 ggtctagctc tagctgtaga aaacatgagg atcacccatg tctgctggac gactgtagaa
     1321 aacatgagga tcacccatgt ctgctgtcta gctgtagaaa acatgaggat cacccatgtc
     1381 tgctggacga ctgtagaaaa catgaggatc acccatgtct gctgtctagc tgtagaaaac
     1441 atgaggatca cccatgtctg ctggacgact gtagaaaaca tgaggatcac ccatgtctgc
     1501 tgtctagctg tagaaaacat gaggatcacc catgtctgct ggacgactgt agaaaacatg
     1561 aggatcaccc atgtctgccg tctagagggc ccgtttaaac ccgctgatca gcctcgactg
     1621 tgccttctag gatcctctag tcagctgacg cgtgctagcg cggccgcatc gataagcttg
     1681 tcgacgatat ctctagagga tcataatcag ccataccaca tttgtagagg ttttacttgc
     1741 tttaaaaaac ctcccacacc tccccctgaa cctgaaacat aaaatgaatg caattgttgt
     1801 tgttaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     1861 cacaaataaa gcattttttt cactgcctcg agcttcctcg ctcactgact cgctgcgctc
     1921 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     1981 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     2041 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     2101 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     2161 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     2221 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct caatgctcac gctgtaggta
     2281 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     2341 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     2401 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     2461 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg
     2521 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     2581 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     2641 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     2701 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     2761 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     2821 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     2881 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     2941 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     3001 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     3061 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     3121 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     3181 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     3241 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     3301 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     3361 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     3421 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     3481 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     3541 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     3601 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     3661 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     3721 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     3781 aggggttccg cgcacatttc ccagatcttg catctcaatt agtcagcaac catagtcccg
     3841 cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat
     3901 cgctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc
     3961 cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct taagcttggc
     4021 aatccggtac tgttggtaaa gccaccatgg aagatgccaa aaacattaag aagggcccag
     4081 cgccattcta cccactcgaa gacgggaccg ccggcgagca gctgcacaaa gccatgaagc
     4141 gctacgccct ggtgcccggc accatcgcct ttaccgacgc acatatcgag gtggacatta
     4201 cctacgccga gtacttcgag atgagcgttc ggctggcaga agctatgaag cgctatgggc
     4261 tgaatacaaa ccatcggatc gtggtgtgca gcgagaatag cttgcagttc ttcatgcccg
     4321 tgttgggtgc cctgttcatc ggtgtggctg tggccccagc taacgacatc tacaacgagc
     4381 gcgagctgct gaacagcatg ggcatcagcc agcccaccgt cgtattcgtg agcaagaaag
     4441 ggctgcaaaa gatcctcaac gtgcaaaaga agctaccgat catacaaaag atcatcatca
     4501 tggatagcaa gaccgactac cagggcttcc aaagcatgta caccttcgtg acttcccatt
     4561 tgccacccgg cttcaacgag tacgacttcg tgcccgagag cttcgaccgg gacaaaacca
     4621 tcgccctgat catgaacagt agtggcagta ccggattgcc caagggcgta gccctaccgc
     4681 accgcaccgc ttgtgtccga ttcagtcatg cccgcgaccc catcttcggc aaccagatca
     4741 tccccgacac cgctatcctc agcgtggtgc catttcacca cggcttcggc atgttcacca
     4801 cgctgggcta cttgatctgc ggctttcggg tcgtgctcat gtaccgcttc gaggaggagc
     4861 tattcttgcg cagcttgcaa gactataaga ttcaatctgc cctgctggtg cccacactat
     4921 ttagcttctt cgctaagagc actctcatcg acaagtacga cctaagcaac ttgcacgaga
     4981 tcgccagcgg cggggcgccg ctcagcaagg aggtaggtga ggccgtggcc aaacgcttcc
     5041 acctaccagg catccgccag ggctacggcc tgacagaaac aaccagcgcc attctgatca
     5101 cccccgaagg ggacgacaag cctggcgcag taggcaaggt ggtgcccttc ttcgaggcta
     5161 aggtggtgga cttggacacc ggtaagacac tgggtgtgaa ccagcgcggc gagctgtgcg
     5221 tccgtggccc catgatcatg agcggctacg ttaacaaccc cgaggctaca aacgctctca
     5281 tcgacaagga cggctggctg cacagcggcg acatcgccta ctgggacgag gacgagcact
     5341 tcttcatcgt ggaccggctg aagagcctga tcaaatacaa gggctaccag gtagccccag
     5401 ccgaactgga gagcatcctg ctgcaacacc ccaacatctt cgacgccggg gtcgccggcc
     5461 tgcccgacga cgatgccggc gagctgcccg ccgcagtcgt cgtgctggaa cacggtaaaa
     5521 ccatgaccga gaaggagatc gtggactatg tggccagcca ggttacaacc gccaagaagc
     5581 tgcgcggtgg tgttgtgttc gtggacgagg tgcctaaagg actgaccggc aagttggacg
     5641 cccgcaagat ccgcgagatt ctcattaagg ccaagaaggg cggcaagatc gccgtgtaat
     5701 aattctagag tcggggcggc cggccgcttc gagcagacat gataagatac attgatgagt
     5761 ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg
     5821 ctattgcttt atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca
     5881 ttcattttat gtttcaggtt cagggggagg tgtgggaggt tttttaaagc aagtaaaacc
     5941 tctacaaatg tggtaaaatc gataaggatc cgaaaagtgc cacctgacgt ctaagaaacc
     6001 attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtcttca
//
