LOCUS       sequence-266130-        5518 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PiggyBac transposon carrying tetR-3xFLAG-HA-2A-Puro under the PGK 
            promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5518)
  AUTHORS   Tsujimura T, Takase O, Yoshikawa M, Sano E, Hayashi M, Hoshi K, 
            Takato T, Toyoda A, Okano H, Hishikawa K
  TITLE     Controlling gene activation by enhancers through a drug-inducible 
            topological insulator.
  JOURNAL   Elife. 2020 May 5;9. pii: 47980. doi: 10.7554/eLife.47980.
  PUBMED    32369019
REFERENCE   2  (bases 1 to 5518)
  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: "Elife."; 
            date: "2020-05-5"; volume: "9. pii: 47980. doi"; pages: " 
            10.7554/eLife.47980"
COMMENT     Sequence Label: pUC57-PB-PGK-tetR-3xFLAG-HA-2A-Puro
FEATURES             Location/Qualifiers
     source          1..5518
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   446..480
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        777..1287
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     1206..1224
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             complement(1437..1448)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             1944..1964
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2073..2138
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             2148..2174
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YPYDVPDYA"
     CDS             2148..2174
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             2205..2258
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     primer_bind     complement(2896..2915)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2950..2969
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   3159..3221
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(3297..3313)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3297..3313)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3310..3332)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3321..3337
                     /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(3345..3351,3352..3369,3370..3375))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3390..3411
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3528..3545)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3699..4287)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3779..3798)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4458..5249,5250..5318))
                     /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     5081..5100
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5319..5423)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5491..5509
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acctcgcgaa
      421 tgcatctaga tatcccaatg attaacccta gaaagatagt ctgcgtaaaa ttgacgcatg
      481 cattcttgaa atattgctct ctctttctaa atagcgcgaa tccgtcgctg tgcatttagg
      541 acatctcagt cgccgcttgg agctcccgtg aggcgtgctt gtcaatgcgg taagtgtcac
      601 tgattttgaa ctataacgac cgcgtgagtc aaaatgacgc atgattatct tttacgtgac
      661 ttttaagatt taactcatac gataattata ttgttatttc atgttctact tacgtgataa
      721 cttattatat atatattttc ttgttataga tatcaagctt atcctcgaaa ttccacgggg
      781 ttggggttgc gccttttcca aggcagccct gggtttgcgc agggacgcgg ctgctctggg
      841 cgtggttccg ggaaacgcag cggcgccgac cctgggtctc gcacattctt cacgtccgtt
      901 cgcagcgtca cccggatctt cgccgctacc cttgtgggcc ccccggcgac gcttcctgct
      961 ccgcccctaa gtcgggaagg ttccttgcgg ttcgcggcgt gccggacgtg acaaacggaa
     1021 gccgcacgtc tcactagtac cctcgcagac ggacagcgcc agggagcaat ggcagcgcgc
     1081 cgaccgcgat gggctgtggc caatagcggc tgctcagcag ggcgcgccga gagcagcggc
     1141 cgggaagggg cggtgcggga ggcggggtgt ggggcggtag tgtgggccct gttcctgccc
     1201 gcgcggtgtt ccgcattctg caagcctccg gagcgcacgt cggcagtcgg ctccctcgtt
     1261 gaccgaatca ccgacctctc tccccaggca tccatctaga tctcgagcag ctgaagctta
     1321 ccatggcaag gctcgacaaa agcaaagtga tcaacagcgc tttggaactg ctgaatgaag
     1381 tagggattga gggcctcaca acacgaaaac tcgcccagaa actgggcgtg gagcagccta
     1441 ccctctattg gcacgtgaag aacaagagag ctctgttgga cgcccttgcc attgagatgc
     1501 tggatagaca ccatacccac ttttgtcctc tggaaggaga gagctggcaa gattttctgc
     1561 ggaataatgc caaatctttt cgctgtgcct tgctgagcca tcgcgacgga gccaaagttc
     1621 atcttgggac ccgacctaca gagaagcagt acgaaacgct ggagaatcag ctcgccttcc
     1681 tgtgccagca agggttttcc ctcgaaaacg cactgtatgc tctgagcgct gtcgggcact
     1741 ttacactcgg ttgcgtgctc gaggatcagg aacatcaagt tgcaaaggag gaacgtgaaa
     1801 caccgacaac cgattctatg cctcccctgt tgcgccaggc aattgagttg tttgaccacc
     1861 agggtgcgga acccgctttc cttttcggcc ttgagctgat catatgcggc cttgaaaagc
     1921 agctgaaatg tgagtccggg tcaccaaaga agaagaggaa ggtggatggc ggaggagcgc
     1981 tgagtccaca acattccgcc gtgactcaag gaagtatcat caagaacaaa gagggcatgg
     2041 acgccaaatc tctgactgct tggtcagcta gcgactacaa agaccatgac ggtgattata
     2101 aagatcatga catcgattac aaggatgacg atgacaaggg agttaactac ccctacgacg
     2161 tgcccgacta cgccttcgaa atcaaaagtt cagtcggttc cggagagggc cggggctctc
     2221 tgctgacctg tggcgacgtg gaggagaacc ccggcccctc catgaccgag tacaagccaa
     2281 ccgtgcgact ggccacgagg gatgacgtcc ccagagccgt tcgtacgctg gctgctgcct
     2341 ttgcggacta tcctgccaca agacacaccg tcgatccgga ccggcatatc gaacgcgtga
     2401 ccgaactgca ggagctgttc ctgacacgag tcggcttgga catagggaag gtatgggtgg
     2461 cagatgatgg tgccgctgtt gccgtctgga caaccccgga aagcgtggaa gcaggagcag
     2521 tgttcgctga gattggaccc agaatggccg agctctctgg gagtaggttg gccgctcaac
     2581 agcagatgga aggcctgctt gccccacatc ggcctaagga gcctgcgtgg tttctggcga
     2641 ctgttggcgt gtcacccgat caccagggta aagggcttgg cagcgctgtg gtgctcccag
     2701 gagtcgaagc agcagagaga gctggtgtac ccgcctttct ggagacttcc gcacctcgca
     2761 acctcccctt ctacgagagg ctgggcttca ccgttactgc cgacgtggag gtgcctgaag
     2821 gaccacggac atggtgcatg actcgcaaac caggggcttg attgtttatt gcagcttata
     2881 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     2941 attctagttg tggtttgtcc aaactcatca atgtatctta tcgataaaag ttttgttact
     3001 ttatagaaga aattttgagt ttttgttttt tttaataaat aaataaacat aaataaattg
     3061 tttgttgaat ttattattag tatgtaagtg taaatataat aaaacttaat atctattcaa
     3121 attaataaat aaacctcgat atacagaccg ataaaacaca tgcgtcaatt ttacacatga
     3181 ttatctttaa cgtacgtcac aatatgatta tctttctagg gttaatctag cattgggata
     3241 tcggatcccg ggcccgtcga ctgcagaggc ctgcatgcaa gcttggcgta atcatggtca
     3301 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     3361 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     3421 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     3481 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     3541 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     3601 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     3661 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     3721 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     3781 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     3841 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     3901 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     3961 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     4021 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     4081 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     4141 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     4201 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     4261 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     4321 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     4381 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     4441 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     4501 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     4561 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     4621 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     4681 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     4741 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     4801 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     4861 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     4921 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     4981 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     5041 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     5101 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     5161 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     5221 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     5281 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     5341 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     5401 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa
     5461 accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtc
//
