LOCUS       sequence-353296-        6031 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  EGFP for the tet-off system.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6031)
  TITLE     Plasmid expressing the monomeric EGFP under the control of the TRE 
            promoter
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6031)
  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            "Unpublished"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: pTRE2-Hyg-EGFP
FEATURES             Location/Qualifiers
     source          1..6031
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    join(15..33,34..56,57..75,76..98,99..117,118..140,141..159,
                     160..182,183..201,202..224,225..243,244..266,267..285)
                     /label=tetracycline response element
                     /bound_moiety="tetracycline repressor TetR"
                     /note="contains seven copies of the tetracycline operator 
                     tetO"
     promoter        318..356
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     353..377
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             join(495..497,498..500,501..1214)
                     /codon_start=1
                     /product="Emerald GFP"
                     /label=EmGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIK
                     VNFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(540..561)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(801..820)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1148..1169
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     intron          1248..1820
                     /label=beta-globin intron
                     /note="intron from rabbit beta-globin gene"
     primer_bind     complement(1952..1971)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2017..2072
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2071..2090)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(2470..2487)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2641..3229)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2721..2740)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3400..4191,4192..4260))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     4023..4042
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4261..4365)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4433..4451
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     polyA_signal    4471..4552
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     4496..4515
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(4669..5694)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             complement(4669..5694)
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     promoter        complement(join(5703..6031,1))
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5717..5852
                     /label=SV40 ori
                     /note="SV40 origin of replication"
ORIGIN
        1 ctcgagttta ccactcccta tcagtgatag agaaaagtga aagtcgagtt taccactccc
       61 tatcagtgat agagaaaagt gaaagtcgag tttaccactc cctatcagtg atagagaaaa
      121 gtgaaagtcg agtttaccac tccctatcag tgatagagaa aagtgaaagt cgagtttacc
      181 actccctatc agtgatagag aaaagtgaaa gtcgagttta ccactcccta tcagtgatag
      241 agaaaagtga aagtcgagtt taccactccc tatcagtgat agagaaaagt gaaagtcgag
      301 ctcggtaccc gggtcgaggt aggcgtgtac ggtgggaggc ctatataagc agagctcgtt
      361 tagtgaaccg tcagatcgcc tggagacgcc atccacgctg ttttgacctc catagaagac
      421 accgggaccg atccagcctc cgcggccccg aattcgagct cggtacccgg ggatcctcta
      481 gtcagctgac gcgtatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc
      541 tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg
      601 gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg
      661 tgccctggcc caccctcgtg accaccttca cctacggcgt gcagtgcttc gcccgctacc
      721 ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg
      781 agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg
      841 agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca
      901 acatcctggg gcacaagctg gagtacaact acaacagcca caaggtctat atcaccgccg
      961 acaagcagaa gaacggcatc aaggtgaact tcaagacccg ccacaacatc gaggacggca
     1021 gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc
     1081 tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc
     1141 gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg
     1201 agctgtacaa gtaagtcgac gatatctcta gagctgagaa cttcagggtg agtttgggga
     1261 cccttgattg ttctttcttt ttcgctattg taaaattcat gttatatgga gggggcaaag
     1321 ttttcagggt gttgtttaga atgggaagat gtcccttgta tcaccatgga ccctcatgat
     1381 aattttgttt ctttcacttt ctactctgtt gacaaccatt gtctcctctt attttctttt
     1441 cattttctgt aactttttcg ttaaacttta gcttgcattt gtaacgaatt tttaaattca
     1501 cttttgttta tttgtcagat tgtaagtact ttctctaatc actttttttt caaggcaatc
     1561 agggtatatt atattgtact tcagcacagt tttagagaac aattgttata attaaatgat
     1621 aaggtagaat atttctgcat ataaattctg gctggcgtgg aaatattctt attggtagaa
     1681 acaactacac cctggtcatc atcctgcctt tctctttatg gttacaatga tatacactgt
     1741 ttgagatgag gataaaatac tctgagtcca aaccgggccc ctctgctaac catgttcatg
     1801 ccttcttctc tttcctacag ctcctgggca acgtgctggt tgttgtgctg tctcatcatt
     1861 ttggcaaaga attcactcct caggtgcagg ctgcctatca gaaggtggtg gctggtgtgg
     1921 ccaatgccct ggctcacaaa taccactgag atctttttcc ctctgccaaa aattatgggg
     1981 acatcatgaa gccccttgag catctgactt ctggctaata aaggaaattt attttcattg
     2041 caatagtgtg ttggaatttt ttgtgtctct cactcggaag gacatatggg agggcaaatc
     2101 atttaaaaca tcagaatgag tatttggttt agagtttggc aacatatgcc catatgctgg
     2161 ctgccatgaa caaaggttgg ctataaagag gtcatcagta tatgaaacag ccccctgctg
     2221 tccattcctt attccataga aaagccttga cttgaggtta gatttttttt atattttgtt
     2281 ttgtgttatt tttttcttta acatccctaa aattttcctt acatgtttta ctagccagat
     2341 ttttcctcct ctcctgacta ctcccagtca tagctgtccc tcttctctta tggagatccc
     2401 tcgactgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     2461 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     2521 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     2581 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     2641 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     2701 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     2761 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     2821 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     2881 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     2941 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     3001 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     3061 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     3121 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     3181 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     3241 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     3301 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     3361 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     3421 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     3481 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     3541 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     3601 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     3661 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     3721 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     3781 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     3841 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     3901 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     3961 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaacacg
     4021 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     4081 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     4141 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     4201 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     4261 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     4321 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     4381 agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg
     4441 tatcacgagg ccctttcgtc ttcactcgag gcagtgaaaa aaatgcttta tttgtgaaat
     4501 ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag ttaacaacaa
     4561 caattgcatt cattttatgt ttcaggttca gggggaggtg tgggaggttt tttaaagcaa
     4621 gtaaaacctc tacaaatgtg gtatggctga ttatgatcct ctagaactct attcctttgc
     4681 cctcggacga gtgctggggc gtcggtttcc actatcggcg agtacttcta cacagccatc
     4741 ggtccagacg gccgcgcttc tgcgggcgat ttgtgtacgc ccgacagtcc cggctccgga
     4801 tcggacgatt gcgtcgcatc gaccctgcgc ccaagctgca tcatcgaaat tgccgtcaac
     4861 caagctctga tagagttggt caagaccaat gcggagcata tacgcccgga gccgcggcga
     4921 tcctgcaagc tccggatgcc tccgctcgaa gtagcgcgtc tgctgctcca tacaagccaa
     4981 ccacggcctc cagaagaaga tgttggcgac ctcgtattgg gaatccccga acatcgcctc
     5041 gctccagtca atgaccgctg ttatgcggcc attgtccgtc aggacattgt tggagccgaa
     5101 atccgcgtgc acgaggtgcc ggacttcggg gcagtcctcg gcccaaagca tcagctcatc
     5161 gagagcctgc gcgacggacg cactgacggt gtcgtccatc acagtttgcc agtgatacac
     5221 atggggatca gcaatcgcgc atatgaaatc acgccatgta gtgtattgac cgattccttg
     5281 cggtccgaat gggccgaacc cgctcgtctg gctaagatcg gccgcagcga tcgcatccat
     5341 ggcctccgcg accggctgca gaacagcggg cagttcggtt tcaggcaggt cttgcaacgt
     5401 gacaccctgt gcacggcggg agatgcaata ggtcaggctc tcgctgaatt ccccaatgtc
     5461 aagcacttcc ggaatcggga gcgcggccga tgcaaagtgc cgataaacat aacgatcttt
     5521 gtagaaacca tcggcgcagc tatttacccg caggacatat ccacgccctc ctacatcgaa
     5581 gctgaaagca cgagattctt cgccctccga gagctgcatc aggtcggaga cgctgtcgaa
     5641 cttttcgatc agaaacttct cgacagacgt cgcggtgagt tcaggctttt tcatggaagc
     5701 tttttgcaaa agcctaggcc tccaaaaaag cctcctcact acttctggaa tagctcagag
     5761 gccgaggcgg cctcggcctc tgcataaata aaaaaaatta gtcagccatg gggcggagaa
     5821 tgggcggaac tgggcggagt taggggcggg atgggcggag ttaggggcgg gactatggtt
     5881 gctgactaat tgagatgcat gctttgcata cttctgcctg ctggggagcc tggggacttt
     5941 ccacacctgg ttgctgacta attgagatgc atgctttgca tacttctgcc tgctggggag
     6001 cctggggact ttccacaccc taactgacac a
//
