LOCUS       sequence-275878-        6591 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tunable and temporal expression control of eGFP (or gene of 
            interest) with constitutive nuclear eGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6591)
  TITLE     Xlone Puro plasmids for inducible gene expression in mammal cells
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6591)
  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            "Unpublished"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: XLone-eGFP NLS (no drug resistant gene)
FEATURES             Location/Qualifiers
     source          1..6591
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        34..245
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      265..274
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             274..297
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             298..1041
                     /codon_start=1
                     /product="modified rtTA protein that binds tightly to 
                     promoters containing the tet operator in the presence of 
                     doxycycline"
                     /label=Tet-On(R) 3G
                     /translation="MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHSCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLKQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     CDS             1051..1107
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             join(1108..1110,1111..1113,1114..1824)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1108..1110,1111..1113,1114..1824)
                     /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             1825..1845
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    1887..1968
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   2144..2206
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(2232..2248)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2232..2248)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2245..2267)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2256..2272
                     /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(2280..2286,2287..2304,2305..2310))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2325..2346
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2463..2480)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2634..3222)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2714..2733)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3393..4184,4185..4253))
                     /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     4016..4035
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4254..4358)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4426..4444
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4482..4504)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4604..4623
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4817..4839
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4831..4848
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4832..4848
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   4859..4893
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    5169..5303
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(join(5493..6203,6204..6206,6207..6209))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(5493..6203,6204..6206,6207..6209))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     promoter        complement(6216..6583)
                     /label=TRE3GS promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G, modified to eliminate
                     binding sites for endogenous mammalian transcription 
                     factors"
     protein_bind    6341..6359
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6377..6395
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6413..6431
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6449..6467
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6485..6503
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6521..6539
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6557..6575
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
ORIGIN
        1 aattccactc gagtggctcc ggtgcccgtc agtgggcaga gcgcacatcg cccacagtcc
       61 ccgagaagtt ggggggaggg gtcggcaatt gaaccggtgc ctagagaagg tggcgcgggg
      121 taaactggga aagtgatgtc gtgtactggc tccgcctttt tcccgagggt gggggagaac
      181 cgtatataag tgcagtagtc gccgtgaacg ttctttttcg caacgggttt gccgccagaa
      241 cacaggtgtc gtgacgcggg atccgccacc atggattaca aagacgatga cgataagatg
      301 tctagactgg acaagagcaa agtcataaac tctgctctgg aattactcaa tggagtcggt
      361 atcgaaggcc tgacgacaag gaaactcgct caaaagctgg gagttgagca gcctaccctg
      421 tactggcacg tgaagaacaa gcgggccctg ctcgatgccc tgccaatcga gatgctggac
      481 aggcatcata cccactcctg ccccctggaa ggcgagtcat ggcaagactt tctgcggaac
      541 aacgccaagt cataccgctg tgctctcctc tcacatcgcg acggggctaa agtgcatctc
      601 ggcacccgcc caacagagaa acagtacgaa accctggaaa atcagctcgc gttcctgtgt
      661 cagcaaggct tctccctgga gaacgcactg tacgctctgt ccgccgtggg ccactttaca
      721 ctgggctgcg tattggagga acaggagcat caagtagcaa aagaggaaag agagacacct
      781 accaccgatt ctatgccccc acttctgaaa caagcaattg agctgttcga ccggcaggga
      841 gccgaacctg ccttcctttt cggcctggaa ctaatcatat gtggcctgga gaaacagcta
      901 aagtgcgaaa gcggcgggcc gaccgacgcc cttgacgatt ttgacttaga catgctccca
      961 gccgatgccc ttgacgactt tgaccttgat atgctgcctg ctgacgctct tgacgatttt
     1021 gaccttgaca tgctccccgg gggcagcgga gccactaact tctccctgtt gaaacaagca
     1081 ggggatgtcg aagagaatcc cgggccaatg gtgagcaagg gcgaggagct gttcaccggg
     1141 gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc
     1201 ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc
     1261 ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc
     1321 ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa
     1381 ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc
     1441 gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc
     1501 aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc
     1561 tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac
     1621 atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac
     1681 ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac
     1741 cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact
     1801 ctcggcatgg acgagctgta caagccaaag aagaagcgta aggtataagt acaagtaaga
     1861 attccgatca tattcaataa cccgttaact tgtttattgc agcttataat ggttacaaat
     1921 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcta aaagttttgt
     1981 tactttatag aagaaatttt gagtttttgt ttttttttaa taaataaata aacataaata
     2041 aattgtttgt tgaatttatt attagtatgt aagtgtaaat ataataaaac ttaatatcta
     2101 ttcaaattaa taaataaacc tcgatataca gaccgataaa acacatgcgt caattttacg
     2161 catgattatc tttaacgtac gtcacaatat gattatcttt ctagggttaa gtcgaccttg
     2221 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     2281 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     2341 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     2401 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     2461 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     2521 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     2581 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     2641 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     2701 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     2761 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     2821 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     2881 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     2941 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     3001 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     3061 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     3121 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     3181 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     3241 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     3301 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     3361 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     3421 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     3481 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     3541 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     3601 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     3661 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     3721 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     3781 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     3841 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     3901 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     3961 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     4021 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     4081 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     4141 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     4201 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     4261 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     4321 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     4381 cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg
     4441 aggccctttc gtctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc
     4501 ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc
     4561 gcgtcagcgg gtgttggcgg gtgtcggggc tggcttaact atgcggcatc agagcagatt
     4621 gtactgagag tgcaccatat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac
     4681 cgcatcaggc gccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg
     4741 gcctcttcgc tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg
     4801 gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggccagtga attcttaacc
     4861 ctagaaagat agtctgcgta aaattgacgc atgcattctt gaaatattgc tctctctttc
     4921 taaatagcgc gaatccgtcg ctgtgcattt aggacatctc agtcgccgct tggagctccc
     4981 gtgaggcgtg cttgtcaatg cggtaagtgt cactgatttt gaactataac gaccgcgtga
     5041 gtcaaaatga cgcatgatta tcttttacgt gacttttaag atttaactca tacgataatt
     5101 atattgttat ttcatgttct acttacgtga taacttatta tatatatatt ttcttgttat
     5161 agatatcaaa cttgtttatt gcagcttata atggttacaa ataaggcaat agcatcacaa
     5221 atttcacaaa taaggcattt ttttcactgc attctagttt tggtttgtcc aaactcatca
     5281 atgtatctta tcatgtctgg atctcaaatc cctcggaagc tgcgcctgtc ttaggttgga
     5341 gtgatacatt tttatcactt ttacccgtct ttggattagg cagtagctct gacggccctc
     5401 ctgtcttagg ttagtgaaaa atgtcactct cttacccgtc attggctgtc cagcttagct
     5461 cgcaggggag gtggtctact agtttagaat tccttgtaca gctcgtccat gccgagagtg
     5521 atcccggcgg cggtcacgaa ctccagcagg accatgtgat cgcgcttctc gttggggtct
     5581 ttgctcaggg cggactgggt gctcaggtag tggttgtcgg gcagcagcac ggggccgtcg
     5641 ccgatggggg tgttctgctg gtagtggtcg gcgagctgca cgctgccgtc ctcgatgttg
     5701 tggcggatct tgaagttcac cttgatgccg ttcttctgct tgtcggccat gatatagacg
     5761 ttgtggctgt tgtagttgta ctccagcttg tgccccagga tgttgccgtc ctccttgaag
     5821 tcgatgccct tcagctcgat gcggttcacc agggtgtcgc cctcgaactt cacctcggcg
     5881 cgggtcttgt agttgccgtc gtccttgaag aagatggtgc gctcctggac gtagccttcg
     5941 ggcatggcgg acttgaagaa gtcgtgctgc ttcatgtggt cggggtagcg gctgaagcac
     6001 tgcacgccgt aggtcagggt ggtcacgagg gtgggccagg gcacgggcag cttgccggtg
     6061 gtgcagatga acttcagggt cagcttgccg taggtggcat cgccctcgcc ctcgccggac
     6121 acgctgaact tgtggccgtt tacgtcgccg tccagctcga ccaggatggg caccaccccg
     6181 gtgaacagct cctcgccctt gctcaccatg gtacctttac gagggtagga agtggtacgg
     6241 aaagttggta taagacaaaa gtgttgtgga attgaagttt actcaaaaaa tcagcactct
     6301 tttataggcg ccctggttta cataagcaaa gcttatacgt tctctatcac tgatagggag
     6361 taaactggat atacgttctc tatcactgat agggagtaaa ctgtagatac gttctctatc
     6421 actgataggg agtaaactgg tcatacgttc tctatcactg atagggagta aactccttat
     6481 acgttctcta tcactgatag ggagtaaagt ctgcatacgt tctctatcac tgatagggag
     6541 taaactcttc atacgttctc tatcactgat agggagtaaa ctcgaggtga t
//
