LOCUS       sequence-328552-        5892 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  eGFP and uL10 ribosomal protein under the TRE3G promoter, Neo 
            resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5892)
  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 5892)
  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-EGFPC-uL10-(Neo)R
FEATURES             Location/Qualifiers
     source          1..5892
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..19
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    37..55
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    73..91
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    109..127
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    145..163
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    181..199
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    217..235
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     280..304
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             join(378..380,381..383,384..1094)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(378..380,381..383,384..1094)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(423..444)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(684..703)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1031..1052
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     intron          2269..2334
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             2464..2484
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    2909..3030
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2946..2965)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3000..3019
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3116..3133)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3287..3875)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3367..3386)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    4204..4251
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     4276..4295
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     CDS             complement(4483..5277)
                     /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(4483..5277)
                     /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(4594..4613)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     5204..5223
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(5312..5669)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5326..5461
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        5478..5669
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     5626..5646
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        complement(5671..5775)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5843..5861
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(5885..5892,1..371)
                     /label=TRE3G promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G"
ORIGIN
        1 tccctatcag tgatagagaa cgtatgaaga gtttactccc tatcagtgat agagaacgta
       61 tgcagacttt actccctatc agtgatagag aacgtataag gagtttactc cctatcagtg
      121 atagagaacg tatgaccagt ttactcccta tcagtgatag agaacgtatc tacagtttac
      181 tccctatcag tgatagagaa cgtatatcca gtttactccc tatcagtgat agagaacgta
      241 taagctttag gcgtgtacgg tgggcgccta taaaagcaga gctcgtttag tgaaccgtca
      301 gatcgcctgg agcaattcca caacactttt gtcttatacc aactttccgt accacttcct
      361 accctcgtaa agtcgacatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca
      421 tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg
      481 agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc
      541 ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct
      601 accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc
      661 aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt
      721 tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg
      781 gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg
      841 ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg
      901 gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc
      961 tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga
     1021 agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg
     1081 acgagctgta caagtccgga ctcagatctc gagctcaagc ttcgaattct atgcccaggg
     1141 aagacagggc gacctggaag tccaactact tccttaagat catccaacta ttggatgatt
     1201 atccgaaatg tttcattgtg ggagcagaca atgtgggctc caagcagatg cagcagatcc
     1261 gcatgtccct tcgcgggaag gctgtggtgc tgatgggcaa gaacaccatg atgcgcaagg
     1321 ccatccgagg gcacctggaa aacaacccag ctctggagaa actgctgcct catatccggg
     1381 ggaatgtggg ctttgtgttc accaaggagg acctcactga gatcagggac atgttgctgg
     1441 ccaataaggt gccagctgct gcccgtgctg gtgccattgc cccatgtgaa gtcactgtgc
     1501 cagcccagaa cactggtctc gggcccgaga agacctcctt tttccaggct ttaggtatca
     1561 ccactaaaat ctccaggggc accattgaaa tcctgagtga tgtgcagctg atcaagactg
     1621 gagacaaagt gggagccagc gaagccacgc tgctgaacat gctcaacatc tcccccttct
     1681 cctttgggct ggtcatccag caggtgttcg acaatggcag catctacaac cctgaagtgc
     1741 ttgatatcac agaggaaact ctgcattctc gcttcctgga gggtgtccgc aatgttgcca
     1801 gtgtctgtct gcagattggc tacccaactg ttgcatcagt accccattct atcatcaacg
     1861 ggtacaaacg agtcctggcc ttgtctgtgg agacggatta caccttccca cttgctgaaa
     1921 aggtcaaggc cttcttggct gatccatctg cctttgtggc tgctgcccct gtggctgctg
     1981 ccaccacagc tgctcctgct gctgctgcag ccccagctaa ggttgaagcc aaggaagagt
     2041 cggaggagtc ggacgaggat atgggatttg gtctctttga ctaaagatct atcgatcggc
     2101 cggatatcac gcgtcatatg gctagcctgc agggatccaa tgtaactgta ttcagcgatg
     2161 acgaaattct tagctattgt aatactctag aggatctttg tgaaggaacc ttacttctgt
     2221 ggtgtgacat aattggacaa actacctaca gagatttaaa gctctaaggt aaatataaaa
     2281 tttttaagtg tataatgtgt taaactactg attctaattg tttgtgtatt ttagattcca
     2341 acctatggaa ctgatgaatg ggagcagtgg tggaatgcct ttaatgagga aaacctgttt
     2401 tgctcagaag aaatgccatc tagtgatgat gaggctactg ctgactctca acattctact
     2461 cctccaaaaa agaagagaaa ggtagaagac cccaaggact ttccttcaga attgctaagt
     2521 tttttgagtc atgctgtgtt tagtaataga actcttgctt gctttgctat ttacaccaca
     2581 aaggaaaaag ctgcactgct atacaagaaa attatggaaa aatattctgt aacctttata
     2641 agtaggcata acagttataa tcataacata ctgttttttc ttactccaca caggcataga
     2701 gtgtctgcta ttaataacta tgctcaaaaa ttgtgtacct ttagcttttt aatttgtaaa
     2761 ggggttaata aggaatattt gatgtatagt gccttgacta gagatcataa tcagccatac
     2821 cacatttgta gaggttttac ttgctttaaa aaacctccca cacctccccc tgaacctgaa
     2881 acataaaatg aatgcaattg ttgttgttaa cttgtttatt gcagcttata atggttacaa
     2941 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
     3001 tggtttgtcc aaactcatca atgtatctta tcatgtctgc ggctctagag ctgcattaat
     3061 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     3121 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     3181 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     3241 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     3301 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     3361 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     3421 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     3481 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     3541 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     3601 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     3661 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     3721 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     3781 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     3841 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     3901 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     3961 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     4021 tatatgagta acctgaggct atggcagggc ctgccgcccc gacgttggct gcgagccctg
     4081 ggccttcacc cgaacttggg gggtggggtg gggaaaagga agaaacgcgg gcgtattggc
     4141 cccaatgggg tctcggtggg gtatcgacag agtgccagcc ctgggaccga accccgcgtt
     4201 tatgaacaaa cgacccaaca ccgtgcgttt tattctgtct ttttattgcc gtcatagcgc
     4261 gggttccttc cggtattgtc tccttccgtg tttcagttag cctcccccta gggtgggcga
     4321 agaactccag catgagatcc ccgcgctgga ggatcatcca gccggcgtcc cggaaaacga
     4381 ttccgaagcc caacctttca tagaaggcgg cggtggaatc gaaatctcgt gatggcaggt
     4441 tgggcgtcgc ttggtcggtc atttcgaacc ccagagtccc gctcagaaga actcgtcaag
     4501 aaggcgatag aaggcgatgc gctgcgaatc gggagcggcg ataccgtaaa gcacgaggaa
     4561 gcggtcagcc cattcgccgc caagctcttc agcaatatca cgggtagcca acgctatgtc
     4621 ctgatagcgg tccgccacac ccagccggcc acagtcgatg aatccagaaa agcggccatt
     4681 ttccaccatg atattcggca agcaggcatc gccatgggtc acgacgagat cctcgccgtc
     4741 gggcatgctc gccttgagcc tggcgaacag ttcggctggc gcgagcccct gatgctcttc
     4801 gtccagatca tcctgatcga caagaccggc ttccatccga gtacgtgctc gctcgatgcg
     4861 atgtttcgct tggtggtcga atgggcaggt agccggatca agcgtatgca gccgccgcat
     4921 tgcatcagcc atgatggata ctttctcggc aggagcaagg tgagatgaca ggagatcctg
     4981 ccccggcact tcgcccaata gcagccagtc ccttcccgct tcagtgacaa cgtcgagcac
     5041 agctgcgcaa ggaacgcccg tcgtggccag ccacgatagc cgcgctgcct cgtcttgcag
     5101 ttcattcagg gcaccggaca ggtcggtctt gacaaaaaga accgggcgcc cctgcgctga
     5161 cagccggaac acggcggcat cagagcagcc gattgtctgt tgtgcccagt catagccgaa
     5221 tagcctctcc acccaagcgg ccggagaacc tgcgtgcaat ccatcttgtt caatcatgcg
     5281 aaacgatcct catcctgtct cttgatcgat ctttgcaaaa gcctaggcct ccaaaaaagc
     5341 ctcctcacta cttctggaat agctcagagg ccgaggcggc ctcggcctct gcataaataa
     5401 aaaaaattag tcagccatgg ggcggagaat gggcggaact gggcggagtt aggggcggga
     5461 tgggcggagt taggggcggg actatggttg ctgactaatt gagatgcatg ctttgcatac
     5521 ttctgcctgc tggggagcct ggggactttc cacacctggt tgctgactaa ttgagatgca
     5581 tgctttgcat acttctgcct gctggggagc ctggggactt tccacaccct aactgacaca
     5641 cattccacag ctggttcttt ccgcctcagg actcttcctt tttcaatatt attgaagcat
     5701 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     5761 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat
     5821 tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc ttcaagaatt
     5881 cctcgagttt ac
//
