LOCUS       sequence-443790-        5500 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  eGFP under the TRE3G promoter, Neo resistance. For cloning GOI.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5500)
  AUTHORS   Michalec-Wawiorka B, Czapinski J, Filipek K, Rulak P, Czerwonka A, 
            Tchorzewski M, Rivero-Muller A
  TITLE     An Improved Vector System for Homogeneous and Stable Gene 
            Regulation.
  JOURNAL   Int J Mol Sci. 2021 May 14;22(10). pii: ijms22105206. doi: 
            10.3390/ijms22105206.
  PUBMED    34069024
REFERENCE   2  (bases 1 to 5500)
  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: "Int J Mol 
            Sci."; date: "2021-05-14"; volume: "22(10). pii: ijms22105206. doi";
            pages: " 10.3390/ijms22105206"
COMMENT     Sequence Label: pTRE3G-EGFP-IRES(Neo)R
FEATURES             Location/Qualifiers
     source          1..5500
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        4..382
                     /label=TRE3G promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G"
     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    84..102
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    120..138
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    156..174
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    192..210
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    228..246
                     /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"
     CDS             join(389..391,392..394,395..1108)
                     /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(389..391,392..394,395..1105)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(434..455)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(695..714)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1042..1063
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    join(1128..1703,1704..1706,1707..1715)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1296..1313)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1523..1542
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     intron          1888..1953
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             2083..2103
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    2528..2649
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2565..2584)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2619..2638
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2735..2752)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2906..3494)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2986..3005)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    3823..3870
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     3895..3914
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     CDS             complement(4102..4896)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             complement(4102..4896)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4213..4232)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4823..4842
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(4931..5288)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4945..5080
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        5097..5288
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     5245..5265
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        complement(5290..5394)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5462..5480
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ctcgagttta ctccctatca gtgatagaga acgtatgaag agtttactcc ctatcagtga
       61 tagagaacgt atgcagactt tactccctat cagtgataga gaacgtataa ggagtttact
      121 ccctatcagt gatagagaac gtatgaccag tttactccct atcagtgata gagaacgtat
      181 ctacagttta ctccctatca gtgatagaga acgtatatcc agtttactcc ctatcagtga
      241 tagagaacgt ataagcttta ggcgtgtacg gtgggcgcct ataaaagcag agctcgttta
      301 gtgaaccgtc agatcgcctg gagcaattcc acaacacttt tgtcttatac caactttccg
      361 taccacttcc taccctcgta aagtcgacat ggtgagcaag ggcgaggagc tgttcaccgg
      421 ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc
      481 cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac
      541 cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg
      601 cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga
      661 aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc
      721 cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt
      781 caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt
      841 ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa
      901 catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga
      961 cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga
     1021 ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac
     1081 tctcggcatg gacgagctgt acaagtaaag atctatcgat cggccggccc ctctccctcc
     1141 ccccccccct aacgttactg gccgaagccg cttggaataa ggccggtgtg cgtttgtcta
     1201 tatgttattt tccaccatat tgccgtcttt tggcaatgtg agggcccgga aacctggccc
     1261 tgtcttcttg acgagcattc ctaggggtct ttcccctctc gccaaaggaa tgcaaggtct
     1321 gttgaatgtc gtgaaggaag cagttcctct ggaagcttct tgaagacaaa caacgtctgt
     1381 agcgaccctt tgcaggcagc ggaacccccc acctggcgac aggtgcctct gcggccaaaa
     1441 gccacgtgta taagatacac ctgcaaaggc ggcacaaccc cagtgccacg ttgtgagttg
     1501 gatagttgtg gaaagagtca aatggctctc ctcaagcgta ttcaacaagg ggctgaagga
     1561 tgcccagaag gtaccccatt gtatgggatc tgatctgggg cctcggtaca catgctttac
     1621 atgtgtttag tcgaggttaa aaaaacgtct aggccccccg aaccacgggg acgtggtttt
     1681 cctttgaaaa acacgatgat aatatggcca caaccgggcc ggatatcacg cgtcatatgg
     1741 ctagcctgca gggatccaat gtaactgtat tcagcgatga cgaaattctt agctattgta
     1801 atactctaga ggatctttgt gaaggaacct tacttctgtg gtgtgacata attggacaaa
     1861 ctacctacag agatttaaag ctctaaggta aatataaaat ttttaagtgt ataatgtgtt
     1921 aaactactga ttctaattgt ttgtgtattt tagattccaa cctatggaac tgatgaatgg
     1981 gagcagtggt ggaatgcctt taatgaggaa aacctgtttt gctcagaaga aatgccatct
     2041 agtgatgatg aggctactgc tgactctcaa cattctactc ctccaaaaaa gaagagaaag
     2101 gtagaagacc ccaaggactt tccttcagaa ttgctaagtt ttttgagtca tgctgtgttt
     2161 agtaatagaa ctcttgcttg ctttgctatt tacaccacaa aggaaaaagc tgcactgcta
     2221 tacaagaaaa ttatggaaaa atattctgta acctttataa gtaggcataa cagttataat
     2281 cataacatac tgttttttct tactccacac aggcatagag tgtctgctat taataactat
     2341 gctcaaaaat tgtgtacctt tagcttttta atttgtaaag gggttaataa ggaatatttg
     2401 atgtatagtg ccttgactag agatcataat cagccatacc acatttgtag aggttttact
     2461 tgctttaaaa aacctcccac acctccccct gaacctgaaa cataaaatga atgcaattgt
     2521 tgttgttaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
     2581 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
     2641 tgtatcttat catgtctgcg gctctagagc tgcattaatg aatcggccaa cgcgcgggga
     2701 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     2761 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     2821 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     2881 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     2941 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     3001 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     3061 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     3121 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     3181 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     3241 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     3301 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     3361 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     3421 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     3481 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     3541 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     3601 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa cctgaggcta
     3661 tggcagggcc tgccgccccg acgttggctg cgagccctgg gccttcaccc gaacttgggg
     3721 ggtggggtgg ggaaaaggaa gaaacgcggg cgtattggcc ccaatggggt ctcggtgggg
     3781 tatcgacaga gtgccagccc tgggaccgaa ccccgcgttt atgaacaaac gacccaacac
     3841 cgtgcgtttt attctgtctt tttattgccg tcatagcgcg ggttccttcc ggtattgtct
     3901 ccttccgtgt ttcagttagc ctccccctag ggtgggcgaa gaactccagc atgagatccc
     3961 cgcgctggag gatcatccag ccggcgtccc ggaaaacgat tccgaagccc aacctttcat
     4021 agaaggcggc ggtggaatcg aaatctcgtg atggcaggtt gggcgtcgct tggtcggtca
     4081 tttcgaaccc cagagtcccg ctcagaagaa ctcgtcaaga aggcgataga aggcgatgcg
     4141 ctgcgaatcg ggagcggcga taccgtaaag cacgaggaag cggtcagccc attcgccgcc
     4201 aagctcttca gcaatatcac gggtagccaa cgctatgtcc tgatagcggt ccgccacacc
     4261 cagccggcca cagtcgatga atccagaaaa gcggccattt tccaccatga tattcggcaa
     4321 gcaggcatcg ccatgggtca cgacgagatc ctcgccgtcg ggcatgctcg ccttgagcct
     4381 ggcgaacagt tcggctggcg cgagcccctg atgctcttcg tccagatcat cctgatcgac
     4441 aagaccggct tccatccgag tacgtgctcg ctcgatgcga tgtttcgctt ggtggtcgaa
     4501 tgggcaggta gccggatcaa gcgtatgcag ccgccgcatt gcatcagcca tgatggatac
     4561 tttctcggca ggagcaaggt gagatgacag gagatcctgc cccggcactt cgcccaatag
     4621 cagccagtcc cttcccgctt cagtgacaac gtcgagcaca gctgcgcaag gaacgcccgt
     4681 cgtggccagc cacgatagcc gcgctgcctc gtcttgcagt tcattcaggg caccggacag
     4741 gtcggtcttg acaaaaagaa ccgggcgccc ctgcgctgac agccggaaca cggcggcatc
     4801 agagcagccg attgtctgtt gtgcccagtc atagccgaat agcctctcca cccaagcggc
     4861 cggagaacct gcgtgcaatc catcttgttc aatcatgcga aacgatcctc atcctgtctc
     4921 ttgatcgatc tttgcaaaag cctaggcctc caaaaaagcc tcctcactac ttctggaata
     4981 gctcagaggc cgaggcggcc tcggcctctg cataaataaa aaaaattagt cagccatggg
     5041 gcggagaatg ggcggaactg ggcggagtta ggggcgggat gggcggagtt aggggcggga
     5101 ctatggttgc tgactaattg agatgcatgc tttgcatact tctgcctgct ggggagcctg
     5161 gggactttcc acacctggtt gctgactaat tgagatgcat gctttgcata cttctgcctg
     5221 ctggggagcc tggggacttt ccacacccta actgacacac attccacagc tggttctttc
     5281 cgcctcagga ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     5341 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     5401 tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa
     5461 aaataggcgt atcacgaggc cctttcgtct tcaagaattc
//
