LOCUS       sequence-251566-        5650 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PiggyBac transposon carrying tetR-KRAB-2A-Puro under the PGK 
            promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5650)
  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 5650)
  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-KRAB-2A-Puro
FEATURES             Location/Qualifiers
     source          1..5650
                     /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             2067..2261
                     /codon_start=1
                     /product="Kruppel-associated box (KRAB) transcriptional 
                     repression domain from the human zinc finger protein ZNF10 
                     (Margolin et al., 1994)"
                     /label=KRAB
                     /translation="RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGY
                     QLTKPDVILRLEKGEEPWLV"
     CDS             2337..2390
                     /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(3028..3047)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3082..3101
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   3291..3353
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(3429..3445)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3429..3445)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3442..3464)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3453..3469
                     /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(3477..3483,3484..3501,3502..3507))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3522..3543
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3660..3677)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3831..4419)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3911..3930)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4590..5381,5382..5450))
                     /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     5213..5232
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5451..5555)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5623..5641
                     /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 tggtcaagga ctcttgtgac cttcaaggac gttttcgtag
     2101 acttcaccag agaggagtgg aaactgctgg acactgcgca gcagatcgtc taccggaatg
     2161 tgatgctgga gaactacaag aacctggtca gcctgggcta tcagctcacg aagcccgatg
     2221 tcatactgag attggagaag ggtgaggaac cctggctggt ggagcgggaa attcaccagg
     2281 aaacccaccc agattccgag actgcattcg aaatcaaaag ttcagtcggt tccggagagg
     2341 gccggggctc tctgctgacc tgtggcgacg tggaggagaa ccccggcccc tccatgaccg
     2401 agtacaagcc aaccgtgcga ctggccacga gggatgacgt ccccagagcc gttcgtacgc
     2461 tggctgctgc ctttgcggac tatcctgcca caagacacac cgtcgatccg gaccggcata
     2521 tcgaacgcgt gaccgaactg caggagctgt tcctgacacg agtcggcttg gacataggga
     2581 aggtatgggt ggcagatgat ggtgccgctg ttgccgtctg gacaaccccg gaaagcgtgg
     2641 aagcaggagc agtgttcgct gagattggac ccagaatggc cgagctctct gggagtaggt
     2701 tggccgctca acagcagatg gaaggcctgc ttgccccaca tcggcctaag gagcctgcgt
     2761 ggtttctggc gactgttggc gtgtcacccg atcaccaggg taaagggctt ggcagcgctg
     2821 tggtgctccc aggagtcgaa gcagcagaga gagctggtgt acccgccttt ctggagactt
     2881 ccgcacctcg caacctcccc ttctacgaga ggctgggctt caccgttact gccgacgtgg
     2941 aggtgcctga aggaccacgg acatggtgca tgactcgcaa accaggggct tgattgttta
     3001 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
     3061 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcgataaa
     3121 agttttgtta ctttatagaa gaaattttga gtttttgttt tttttaataa ataaataaac
     3181 ataaataaat tgtttgttga atttattatt agtatgtaag tgtaaatata ataaaactta
     3241 atatctattc aaattaataa ataaacctcg atatacagac cgataaaaca catgcgtcaa
     3301 ttttacacat gattatcttt aacgtacgtc acaatatgat tatctttcta gggttaatct
     3361 agcattggga tatcggatcc cgggcccgtc gactgcagag gcctgcatgc aagcttggcg
     3421 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     3481 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     3541 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     3601 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     3661 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     3721 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     3781 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     3841 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     3901 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     3961 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     4021 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     4081 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     4141 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     4201 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     4261 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     4321 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     4381 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     4441 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     4501 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     4561 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     4621 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     4681 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     4741 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     4801 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     4861 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     4921 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     4981 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     5041 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     5101 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     5161 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     5221 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     5281 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     5341 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     5401 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     5461 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     5521 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     5581 gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg
     5641 ccctttcgtc
//
