LOCUS       sequence-183628-        6327 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tunable and temporal expression control of GFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6327)
  AUTHORS   Randolph LN, Bao X, Zhou C, Lian X
  TITLE     An all-in-one, Tet-On 3G inducible PiggyBac system for human 
            pluripotent stem cells and derivatives.
  JOURNAL   Sci Rep. 2017 May 8;7(1):1549. doi: 10.1038/s41598-017-01684-6.
  PUBMED    28484230
REFERENCE   2  (bases 1 to 6327)
  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: "Sci Rep."; 
            date: "2017-05-8"; volume: "7(1):1549. doi"; pages: " 
            10.1038/s41598-017-01684-6"
COMMENT     Sequence Label: XLone-GFP
FEATURES             Location/Qualifiers
     source          1..6327
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        9..376
                     /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    17..35
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    53..71
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    89..107
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    125..143
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    161..179
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    197..215
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    233..251
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             join(383..385,386..388,389..1099)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(383..385,386..388,389..1099)
                     /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(428..449)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(689..708)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1036..1057
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1289..1423
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   1699..1733
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(1744..1761)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1744..1760)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1753..1775)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(1969..1988)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2088..2110
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2148..2166)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2234..2338
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2339..2407,2408..3199)
                     /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     complement(2557..2576)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3370..3958
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3859..3878
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4112..4129
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4246..4267
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4282..4287,4288..4305,4306..4312)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4320..4336
                     /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)."
     primer_bind     4325..4347
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4344..4360
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4344..4360
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   4386..4448
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    4624..4705
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(4822..5220)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             complement(5221..5277)
                     /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             complement(5287..6030)
                     /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             complement(6031..6054)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      6054..6063
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6083..6294)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
ORIGIN
        1 atcacctcga gtttactccc tatcagtgat agagaacgta tgaagagttt actccctatc
       61 agtgatagag aacgtatgca gactttactc cctatcagtg atagagaacg tataaggagt
      121 ttactcccta tcagtgatag agaacgtatg accagtttac tccctatcag tgatagagaa
      181 cgtatctaca gtttactccc tatcagtgat agagaacgta tatccagttt actccctatc
      241 agtgatagag aacgtataag ctttgcttat gtaaaccagg gcgcctataa aagagtgctg
      301 attttttgag taaacttcaa ttccacaaca cttttgtctt ataccaactt tccgtaccac
      361 ttcctaccct cgtaaaggta ccatggtgag caagggcgag gagctgttca ccggggtggt
      421 gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga
      481 gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa
      541 gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag
      601 ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta
      661 cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt
      721 gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga
      781 ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat
      841 catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga
      901 ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc
      961 cgtgctgctg cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa
     1021 cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg
     1081 catggacgag ctgtacaagg aattctaaac tagtagacca cctcccctgc gagctaagct
     1141 ggacagccaa tgacgggtaa gagagtgaca tttttcacta acctaagaca ggagggccgt
     1201 cagagctact gcctaatcca aagacgggta aaagtgataa aaatgtatca ctccaaccta
     1261 agacaggcgc agcttccgag ggatttgaga tccagacatg ataagataca ttgatgagtt
     1321 tggacaaacc aaaactagaa tgcagtgaaa aaaatgcctt atttgtgaaa tttgtgatgc
     1381 tattgcctta tttgtaacca ttataagctg caataaacaa gtttgatatc tataacaaga
     1441 aaatatatat ataataagtt atcacgtaag tagaacatga aataacaata taattatcgt
     1501 atgagttaaa tcttaaaagt cacgtaaaag ataatcatgc gtcattttga ctcacgcggt
     1561 cgttatagtt caaaatcagt gacacttacc gcattgacaa gcacgcctca cgggagctcc
     1621 aagcggcgac tgagatgtcc taaatgcaca gcgacggatt cgcgctattt agaaagagag
     1681 agcaatattt caagaatgca tgcgtcaatt ttacgcagac tatctttcta gggttaagaa
     1741 ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa
     1801 tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga
     1861 tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg cgcctgatgc ggtattttct
     1921 ccttacgcat ctgtgcggta tttcacaccg catatggtgc actctcagta caatctgctc
     1981 tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg
     2041 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     2101 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg
     2161 cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt
     2221 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     2281 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
     2341 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     2401 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     2461 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     2521 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     2581 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     2641 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     2701 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     2761 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     2821 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     2881 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     2941 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     3001 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
     3061 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     3121 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     3181 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     3241 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     3301 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     3361 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     3421 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     3481 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     3541 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     3601 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     3661 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     3721 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     3781 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     3841 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     3901 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     3961 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     4021 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     4081 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
     4141 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
     4201 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct
     4261 cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat
     4321 tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg ccaaggtcga
     4381 cttaacccta gaaagataat catattgtga cgtacgttaa agataatcat gcgtaaaatt
     4441 gacgcatgtg ttttatcggt ctgtatatcg aggtttattt attaatttga atagatatta
     4501 agttttatta tatttacact tacatactaa taataaattc aacaaacaat ttatttatgt
     4561 ttatttattt attaaaaaaa aacaaaaact caaaatttct tctataaagt aacaaaactt
     4621 ttagcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac
     4681 cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta tgtttcaggt
     4741 tcagggggag gtgtgggagg ttttttaaag caagtaaaac ctctacaaat gtggtatggc
     4801 tgattatgat cctctggaga tttagccctc ccacacataa ccagagggca gcaattcacg
     4861 aatcccaact gccgtcggct gtccatcact gtccttcact atggctttga tcccaggatg
     4921 cagatcgaga agcacctgtc ggcaccgtcc gcaggggctc aagatgcccc tgttctcatt
     4981 tccgatcgcg acgatacaag tcaggttgcc agctgccgca gcagcagcag tgcccagcac
     5041 cacgagttct gcacaaggtc ccccagtaaa atgatataca ttgacaccag tgaagatgcg
     5101 gccgtcgcta gagagagctg cgctggcgac gctgtagtct tcagagatgg ggatgctgtt
     5161 gattgtagcc gttgctcttt caatgagggt ggattcttct tgagacaaag gcttggccat
     5221 ggggccgggg ttctcctcca cgtcgccggc ctgcttcagc aggctgaagt tggtggcgcc
     5281 gctgcccccg gggagcatgt caaggtcaaa atcgtcaaga gcgtcagcag gcagcatatc
     5341 aaggtcaaag tcgtcaaggg catcggctgg gagcatgtct aagtcaaaat cgtcaagggc
     5401 gtcggtcggc ccgccgcttt cgcactttag ctgtttctcc aggccacata tgattagttc
     5461 caggccgaaa aggaaggcag gttcggctcc ctgccggtcg aacagctcaa ttgcttgttt
     5521 cagaagtggg ggcatagaat cggtggtagg tgtctctctt tcctcttttg ctacttgatg
     5581 ctcctgttcc tccaatacgc agcccagtgt aaagtggccc acggcggaca gagcgtacag
     5641 tgcgttctcc agggagaagc cttgctgaca caggaacgcg agctgatttt ccagggtttc
     5701 gtactgtttc tctgttgggc gggtgccgag atgcacttta gccccgtcgc gatgtgagag
     5761 gagagcacag cggtatgact tggcgttgtt ccgcagaaag tcttgccatg actcgccttc
     5821 cagggggcag gagtgggtat gatgcctgtc cagcatctcg attggcaggg catcgagcag
     5881 ggcccgcttg ttcttcacgt gccagtacag ggtaggctgc tcaactccca gcttttgagc
     5941 gagtttcctt gtcgtcaggc cttcgatacc gactccattg agtaattcca gagcagagtt
     6001 tatgactttg ctcttgtcca gtctagacat cttatcgtca tcgtctttgt aatccatggt
     6061 ggcggatccc gcgtcacgac acctgtgttc tggcggcaaa cccgttgcga aaaagaacgt
     6121 tcacggcgac tactgcactt atatacggtt ctcccccacc ctcgggaaaa aggcggagcc
     6181 agtacacgac atcactttcc cagtttaccc cgcgccacct tctctaggca ccggttcaat
     6241 tgccgacccc tccccccaac ttctcgggga ctgtgggcga tgtgcgctct gcccactgac
     6301 gggcaccgga gccactcgag tggaatt
//
