LOCUS       sequence-239656-        6714 bp    DNA     circular SYN 15-APR-2025
DEFINITION  piggyBac-based Dox-inducible expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6714)
  AUTHORS   Adachi K, Kopp W, Wu G, Heising S, Greber B, Stehling M, 
            Arauzo-Bravo MJ, Boerno ST, Timmermann B, Vingron M, Scholer HR
  TITLE     Esrrb Unlocks Silenced Enhancers for Reprogramming to Naive 
            Pluripotency.
  JOURNAL   Cell Stem Cell. 2018 Aug 2;23(2):266-275.e6. doi: 
            10.1016/j.stem.2018.05.020. Epub 2018 Jun 14.
  PUBMED    29910149
REFERENCE   2  (bases 1 to 6714)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.stem.2018.05.020"; journalName: "Cell Stem Cell"; date: 
            "2018-08-2- 2"; volume: "23"; issue: "2"; pages: "266-275.e6"
COMMENT     Sequence Label: KA0718_pPB-TREtight-cHA-IRESVenus
FEATURES             Location/Qualifiers
     source          1..6714
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     623..642
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        623..641
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   660..694
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        1044..1358
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    1052..1070
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1088..1106
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1123..1141
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1159..1177
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1195..1213
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1230..1248
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1266..1284
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     1331..1355
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          1402..1974
                     /label=beta-globin intron
                     /note="intron from rabbit beta-globin gene"
     CDS             2062..2088
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     misc_feature    join(2131..2705,2706..2708,2709..2717)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2298..2315)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2525..2544
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(2718..2720,2721..2723,2724..3437)
                     /codon_start=1
                     /product="yellow fluorescent protein (YFP) with fast and 
                     efficient maturation (Nagai et al., 2002)"
                     /label=Venus
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2763..2784)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3024..3043)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3371..3392
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3822..3839)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3828..4052
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4099..4115)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4123..4139
                     /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(4147..4153,4154..4171,4172..4177))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4192..4213
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     repeat_region   4440..4502
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        complement(4526..4544)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4565..4581)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4589..4605
                     /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(4613..4619,4620..4637,4638..4643))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4658..4679
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4796..4813)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4967..5555)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5047..5066)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5726..6517,6518..6586))
                     /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     6349..6368
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6587..6691)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgaat tgtaatacga ctcactatag ggcgaattgg gtaccttaac
      661 cctagaaaga tagtctgcgt aaaattgacg catgcattct tgaaatattg ctctctcttt
      721 ctaaatagcg cgaatccgtc gctgtgcatt taggacatct cagtcgccgc ttggagctcc
      781 cgtgaggcgt gcttgtcaat gaggtaagtg tcactgattt tgaactataa cgaccgcgtg
      841 agtcaaaatg acgcatgatt atcttttacg tgacttttaa gatttaactc atacgataat
      901 tatattgtta tttcatgttc tacttacgtg ataacttatt atatatatat tttcttgtta
      961 tagatatcat aatttaaaca agcaaaacca aattaagggc cagctcattc ctcccactca
     1021 tgatctatag atctggatcc gtcgagttta ctccctatca gtgatagaga acgtatgtcg
     1081 agtttactcc ctatcagtga tagagaacga tgtcgagttt actccctatc agtgatagag
     1141 aacgtatgtc gagtttactc cctatcagtg atagagaacg tatgtcgagt ttactcccta
     1201 tcagtgatag agaacgtatg tcgagtttat ccctatcagt gatagagaac gtatgtcgag
     1261 tttactccct atcagtgata gagaacgtat gtcgaggtag gcgtgtacgg tgggaggcct
     1321 atataagcag agctcgttta gtgaaccgtc agatcgcctg gagaattcga gctcggtacc
     1381 cgggatcctg agaacttcag ggtgagtttg gggacccttg attgttcttt ctttttcgct
     1441 attgtaaaat tcatgttata tggagggggc aaagttttca gggtgttgtt tagaatggga
     1501 agatgtccct tgtatcacca tggaccctca tgataatttt gtttctttca ctttctactc
     1561 tgttgacaac cattgtctcc tcttattttc ttttcatttt ctgtaacttt ttcgttaaac
     1621 tttagcttgc atttgtaacg aatttttaaa ttcacttttg tttatttgtc agattgtaag
     1681 tactttctct aatcactttt ttttcaaggc aatcagggta tattatattg tacttcagca
     1741 cagttttaga gaacaattgt tataattaaa tgataaggta gaatatttct gcatataaat
     1801 tctggctggc gtggaaatat tcttattggt agaaacaact acaccctggt catcatcctg
     1861 cctttctctt tatggttaca atgatataca ctgtttgaga tgaggataaa atactctgag
     1921 tccaaaccgg gcccctctgc taaccatgtt catgccttct tctctttcct acagctcctg
     1981 ggcaacgtgc tggttgttgt gctgtctcat cattttggca aagaattcct cgagatctca
     2041 cgcgtgacta gtgccatggc ctacccatac gacgtgccag actacgccta gcggccgctc
     2101 gataagcttg atatcgaatt ccgccccccc cccctctccc tccccccccc ctaacgttac
     2161 tggccgaagc cgcttggaat aaggccggtg tgcgtttgtc tatatgttat tttccaccat
     2221 attgccgtct tttggcaatg tgagggcccg gaaacctggc cctgtcttct tgacgagcat
     2281 tcctaggggt ctttcccctc tcgccaaagg aatgcaaggt ctgttgaatg tcgtgaagga
     2341 agcagttcct ctggaagctt cttgaagaca aacaacgtct gtagcgaccc tttgcaggca
     2401 gcggaacccc ccacctggcg acaggtgcct ctgcggccaa aagccacgtg tataagatac
     2461 acctgcaaag gcggcacaac cccagtgcca cgttgtgagt tggatagttg tggaaagagt
     2521 caaatggctc tcctcaagcg tattcaacaa ggggctgaag gatgcccaga aggtacccca
     2581 ttgtatggga tctgatctgg ggcctcggtg cacatgcttt acatgtgttt agtcgaggtt
     2641 aaaaaaacgt ctaggccccc cgaaccacgg ggacgtggtt ttcctttgaa aaacacgatg
     2701 ataatatggc cacaaccatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca
     2761 tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg
     2821 agggcgatgc cacctacggc aagctgaccc tgaagctgat ctgcaccacc ggcaagctgc
     2881 ccgtgccctg gcccaccctc gtgaccaccc tgggctacgg cctgcagtgc ttcgcccgct
     2941 accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc
     3001 aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt
     3061 tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg
     3121 gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcaccg
     3181 ccgacaagca gaagaacggc atcaaggcca acttcaagat ccgccacaac atcgaggacg
     3241 gcggcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc
     3301 tgctgcccga caaccactac ctgagctacc agtccgccct gagcaaagac cccaacgaga
     3361 agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg
     3421 acgagctgta caagtaaagc ggccagctta tcgactcgcc gatagtggaa accgacgccc
     3481 cagcactcgt ccgagggcaa aggaatagtc gagaaattga tgatctatta aacaataaag
     3541 atgtccacat ggaagttttt cctgtcatac tttgttaaga agggtgagaa cagagtacct
     3601 acattttgaa tggaaggatt ggagctacgg gggtgggggt ggggtgggat tagataaatg
     3661 cctgctcttt actgaaggct ctttactatt gctttatgat aatgtttcat agttggatat
     3721 cataatttaa acaagcaaaa ccaaattaag ggccagctca ttcctcccac tcatgatcta
     3781 tagatcctaa gcttatcgat gatctagagc tcgctgatca gcctcgactg tgccttctag
     3841 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac
     3901 tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca
     3961 ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag
     4021 caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa cctgcagccc
     4081 aagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     4141 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     4201 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     4261 ccagcggatc ctttgttact ttatagaaga aattttgagt ttttgttttt ttttaataaa
     4321 taaataaaca taaataaatt gtttgttgaa tttattatta gtatgtaagt gtaaatataa
     4381 taaaacttaa tatctattca aattaataaa taaacctcga tatacagacc gataaaacac
     4441 atgcgtcaat tttacgcatg attatcttta acgtacgtca caatatgatt atctttctag
     4501 ggttaaagag ctccagcttt tgttcccttt agtgagggtt aatttcgagc ttggcgtaat
     4561 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     4621 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
     4681 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     4741 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     4801 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     4861 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     4921 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     4981 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     5041 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     5101 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     5161 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     5221 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     5281 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     5341 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     5401 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     5461 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     5521 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     5581 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     5641 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     5701 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     5761 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     5821 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     5881 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     5941 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     6001 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     6061 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     6121 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     6181 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     6241 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     6301 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     6361 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     6421 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     6481 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacggga aggcaaaatg
     6541 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     6601 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     6661 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccac
//
