LOCUS       sequence-311048-        6661 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  PiggyBac backbone, CAG promoter, Hygromycin selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 6661)
  AUTHORS   Betschinger J, Nichols J, Dietmann S, Corrin PD, Paddison PJ, Smith 
            A
  TITLE     Exit from pluripotency is gated by intracellular redistribution of 
            the bHLH transcription factor Tfe3.
  JOURNAL   Cell. 2013 Apr 11;153(2):335-47. doi: 10.1016/j.cell.2013.03.012.
  PUBMED    23582324
REFERENCE   2  (bases 1 to 6661)
  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"; doi: 
            "10.1016/j.cell.2013.03"; journalName: "Cell"; date: "2013-04-11- 
            11"; volume: "153"; issue: "2"; pages: "335-47"
COMMENT     Sequence Label: pPB-CAG-empty-pgk-hph
FEATURES             Location/Qualifiers
     source          1..6661
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     primer_bind     326..348
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     410..429
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     primer_bind     complement(490..506)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    514..530
                     /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(538..544,545..562,563..568))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    583..604
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(664..681)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    670..894
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    971..1004
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     repeat_region   1175..1237
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        complement(1261..1279)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1300..1316)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1324..1340
                     /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(1348..1354,1355..1372,1373..1378))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1393..1414
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1531..1548)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1702..2290)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1782..1801)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2461..3252,3253..3321))
                     /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     3084..3103
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3322..3426)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3452..3907)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3589..3610)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3801..3820
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4034..4056
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4048..4065
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4049..4065
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4072..4091
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4072..4090
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   4109..4143
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     CDS             complement(4625..5650)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             complement(4625..5650)
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     promoter        complement(5669..6168)
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(5717..5736)
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     primer_bind     6110..6133
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     protein_bind    6191..6224
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     enhancer        6240..6619
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 ggggggggct sgggggaggg gcgcggcggc ccccggagcg ccggcggctg tcgaggcgcg
       61 gcgagccgca gccattgcct tttatggtaa tcgtgcgaga gggcgcaggg acttcctttg
      121 tcccaaatct gtgcggagcc gaaatctggg aggcgccgcc gcaccccctc tagcgggcgc
      181 ggggcgaagc ggtgcggcgc cggcaggaag gaaatgggcg gggagggcct tcgtgcgtcg
      241 ccgcgccgcc gtccccttct ccctctccag cctcggggct gtccgcgggg ggacggctgc
      301 cttcgggggg gacggggcag ggcggggttc ggcttctggc gtgtgaccgg cggctctaga
      361 gcctctgcta accatgttca tgccttcttc tttttcctac agctcctggg caacgtgctg
      421 gttattgtgc tgtctcatca ttttggcaaa gaattcctcg acggtatcga taagcttggc
      481 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
      541 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
      601 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagcggat
      661 cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt
      721 ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat
      781 cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg
      841 gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg
      901 aggcggaaag aaccagctgg ggctcgagat ccactagttc tagcctcgag gctagagcgg
      961 ccgcgtcgag ataacttcgt ataatgtatg ctatacgaag ttatgttttg ttactttata
     1021 gaagaaattt tgagtttttg ttttttttta ataaataaat aaacataaat aaattgtttg
     1081 ttgaatttat tattagtatg taagtgtaaa tataataaaa cttaatatct attcaaatta
     1141 ataaataaac ctcgatatac agaccgataa aacacatgcg tcaattttac gcatgattat
     1201 ctttaacgta cgtcacaata tgattatctt tctagggtta aagagctcca gcttttgttc
     1261 cctttagtga gggttaattt cgagcttggc gtaatcatgg tcatagctgt ttcctgtgtg
     1321 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
     1381 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
     1441 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     1501 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     1561 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     1621 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     1681 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     1741 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     1801 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     1861 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     1921 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     1981 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     2041 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     2101 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     2161 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     2221 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     2281 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     2341 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     2401 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     2461 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     2521 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     2581 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     2641 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     2701 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     2761 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     2821 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     2881 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     2941 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     3001 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     3061 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     3121 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     3181 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     3241 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     3301 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     3361 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     3421 tccgcgcaca tttccccgaa aagtgccacc taaattgtaa gcgttaatat tttgttaaaa
     3481 ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa
     3541 atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac
     3601 aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag
     3661 ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt
     3721 aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg
     3781 gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca
     3841 agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag
     3901 ggcgcgtccc attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc
     3961 tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta
     4021 acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt gtaatacgac
     4081 tcactatagg gcgaattggg taccttaacc ctagaaagat agtctgcgta aaattgacgc
     4141 atgcattctt gaaatattgc tctctctttc taaatagcgc gaatccgtcg ctgtgcattt
     4201 aggacatctc agtcgccgct tggagctccc gtgaggcgtg cttgtcaatg cggtaagtgt
     4261 cactgatttt gaactataac gaccgcgtga gtcaaaatga cgcatgatta tcttttacgt
     4321 gacttttaag atttaactca tacgataatt atattgttat ttcatgttct acttacgtga
     4381 taacttatta tatatatatt ttcttgttat agatatccaa ctatgaaaca ttatcataaa
     4441 gcaatagtaa agagccttca gtaaagagca ggcatttatc taatcccacc ccacccccac
     4501 ccccgtagct ccaatccttc cattcaaaat gtaggtactc tgttctcacc cttcttaaca
     4561 aagtatgaca ggaaaaactt ccatgtggac atctttattg tttaatagat catcaatttc
     4621 tcgactattc ctttgccctc ggacgagtgc tggggcgtcg gtttccacta tcggcgagta
     4681 cttctacaca gccatcggtc cagacggccg cgcttctgcg ggcgatttgt gtacgcccga
     4741 cagtcccggc tccggatcgg acgattgcgt cgcatcgacc ctgcgcccaa gctgcatcat
     4801 cgaaattgcc gtcaaccaag ctctgataga gttggtcaag accaatgcgg agcatatacg
     4861 cccggagccg cggcgatcct gcaagctccg gatgcctccg ctcgaagtag cgcgtctgct
     4921 gctccataca agccaaccac ggcctccaga agaagatgtt ggcgacctcg tattgggaat
     4981 ccccgaacat cgcctcgctc cagtcaatga ccgctgttat gcggccattg tccgtcagga
     5041 cattgttgga gccgaaatcc gcgtgcacga ggtgccggac ttcggggcag tcctcggccc
     5101 aaagcatcag ctcatcgaga gcctgcgcga cggacgcact gacggtgtcg tccatcacag
     5161 tttgccagtg atacacatgg ggatcagcaa tcgcgcatat gaaatcacgc catgtagtgt
     5221 attgaccgat tccttgcggt ccgaatgggc cgaacccgct cgtctggcta agatcggccg
     5281 cagcgatcgc atccatggcc tccgcgaccg gctgcagaac agcgggcagt tcggtttcag
     5341 gcaggtcttg caacgtgaca ccctgtgcac ggcgggagat gcaataggtc aggctctcgc
     5401 tgaattcccc aatgtcaagc acttccggaa tcgggagcgc ggccgatgca aagtgccgat
     5461 aaacataacg atctttgtag aaaccatcgg cgcagctatt tacccgcagg acatatccac
     5521 gccctcctac atcgaagctg aaagcacgag attcttcgcc ctccgagagc tgcatcaggt
     5581 cggagacgct gtcgaacttt tcgatcagaa acttctcgac agacgtcgcg gtgagttcag
     5641 gctttttcat ggtggcggct ggatcggtcg aaaggcccgg agatgaggaa gaggagaaca
     5701 gcgcggcaga cgtgcgcttt tgaagcgtgc agaatgccgg gcctccggag gaccttcggg
     5761 cgcccgcccc gcccctgagc ccgcccctga gcccgccccc ggacccaccc cttcccagcc
     5821 tctgagccca gaaagcgaag gagcaaagct gctattggcc gctgccccaa aggcctaccc
     5881 gcttccattg ctcagcggtg ctgtccatct gcacgagact agtgagacgt gctacttcca
     5941 tttgtcacgt cctgcacgac gcgagctgcg gggcgggggg gaacttcctg actaggggag
     6001 gagtagaagg tggcgcgaag gggccaccaa agaacggagc cggttggcgc ctaccggtgg
     6061 atgtggaatg tgtgcgaggc cagaggccac ttgtgtagcg ccaagtgccc agcggggctg
     6121 ctaaagcgca tgctccagac tgccttggga aaagcgcctc ccctacccgg tagaattcat
     6181 cgatgatatc ataacttcgt ataatgtatg ctatacgaag ttatgcggcc ctcgaggtcg
     6241 acattgatta ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc
     6301 atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa
     6361 cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac
     6421 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca
     6481 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg
     6541 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt
     6601 agtcatcgct attaccatgg tcgaggtgag ccccacgttc tgcttcactc tccccatctc
     6661 c
//
